prof. azaprof. aza

Reference: Reference: Gareth ThomasGareth Thomas

Week 11, 12,13Week 11, 12,13

prof. azaprof. aza

1. Introduction1. Introduction

•The nucleic acids are the The nucleic acids are the compounds that are compounds that are responsible responsible for the storage and transmission for the storage and transmission of the genetic information that of the genetic information that controls controls the growth, function and the growth, function and reproduction of all types of cellsreproduction of all types of cells..

•They are classified into two general They are classified into two general types: types: thethe deoxyribonucleic acids deoxyribonucleic acids (DNA), whose structures contain (DNA), whose structures contain the sugar residue the sugar residue β-β-D-deoxyribose; D-deoxyribose; and and the ribonucleic acids the ribonucleic acids (RNA), (RNA), whose structures contain the sugar whose structures contain the sugar residue residue β -β -D-ribose (Figure 10.1).D-ribose (Figure 10.1).

prof. azaprof. aza

prof. azaprof. aza

Figure 1. The structures of ββ -D-deoxyribose and ββ -D-ribose.

prof. azaprof. aza

nucleotide consists of a purine or nucleotide consists of a purine or pyrimidine basepyrimidine base

• Both types of nucleic acids are Both types of nucleic acids are polymers based on a repeating polymers based on a repeating structural unit known as a nucleotide structural unit known as a nucleotide (Figure 10.2). These nucleotides form (Figure 10.2). These nucleotides form long single-chain polymer molecules long single-chain polymer molecules in both DNA and RNA. in both DNA and RNA.

• Each Each nucleotide consists of a purine nucleotide consists of a purine or pyrimidine baseor pyrimidine base bonded to the 1’ bonded to the 1’ carbon atom of a sugar residue by a carbon atom of a sugar residue by a ββ -N-glycosidic link (Figure 10.3). -N-glycosidic link (Figure 10.3).

•These base-sugar subunits, These base-sugar subunits, which are which are known as nucleosidesknown as nucleosides, , are linked thare linked thrrough the 3’ and 5’ ough the 3’ and 5’ carbons of their sugar residues carbons of their sugar residues by phosphate units to form the by phosphate units to form the polymer chain.polymer chain.

prof. azaprof. aza

prof. azaprof. aza

Figure 2. The general structures of (a) nucleotides and (b) a schematic representation of a section of a nucleic acid chain.

prof. azaprof. aza

Figure 3. Examples of the structures of some of the nucleosides found in RNA. The ββ -N-glycosidic link is shaded The corresponding nucleosides in DNA are based on deoxyribose and use the same name but with the prefix deoxy.

prof. azaprof. aza

22.. Deoxyribonucleic Acids (DNA) Deoxyribonucleic Acids (DNA)

•DNA occurs in the nuclei of cells DNA occurs in the nuclei of cells in the form of a very compact in the form of a very compact DNA protein complex called DNA protein complex called chromatinchromatin. The protein in . The protein in chromatin consists mainly of chromatin consists mainly of histones, a family of relatively histones, a family of relatively small positively charged small positively charged proteins. proteins.

•The DNA is coiled twice around The DNA is coiled twice around as octomer of histone molecules as octomer of histone molecules with a ninth histone molecule with a ninth histone molecule attached to the exterior of these attached to the exterior of these minimini coils to form a structure like coils to form a structure like a row of heads spaced along a a row of heads spaced along a string (Figure 10.4). string (Figure 10.4).

prof. azaprof. aza

• This ‘string of beads’ is coiled and This ‘string of beads’ is coiled and twisted into compact structures known twisted into compact structures known as miniband units, which form the basis as miniband units, which form the basis of the structures of chromosomes. of the structures of chromosomes. Chromosomes are the structures that Chromosomes are the structures that form duplicates during cell division in form duplicates during cell division in order to transfer the genetic order to transfer the genetic information of the old cell to the two information of the old cell to the two new cells.new cells.

prof. azaprof. aza

prof. azaprof. aza

2.1 Structure2.1 StructureDNA molecules are large with relative molecular masses up to one trillion. The principal bases found in their structures are adenine (A), thymine (T), guanine (G) and cytosine (C), although derivatives of these bases are found in some DNA molecules (Figure 10.5). Those bases with an oxygen function have been shown to exist in their keto form.

• Figure 4. The ‘string of heads’ structure Figure 4. The ‘string of heads’ structure of chromatin. The DNA strand is of chromatin. The DNA strand is rround ound twice around each histone octomer. A twice around each histone octomer. A ninth histone molecule is bound to the ninth histone molecule is bound to the exterior surface of the coil.exterior surface of the coil.

prof. azaprof. aza

prof. azaprof. aza

DNA-binding DNA-binding proteinsproteins

Interaction of DNA with histones (shown in white, top). These proteins' basic amino acids (below left, blue) bind to the acidic phosphate groups on DNA (below right, red).

prof. azaprof. aza

Figure 5. The purine and pyrimidine bases found in DNA. The numbering is the same or each type of ring system.

prof. azaprof. aza

Structures of the four bases found in DNA and the nucleotide adenosine monophosphate.

AdenineGuanine

ThymineCytosine

Adenosine monophosphate

prof. azaprof. aza

•Chargaff showed that the molar Chargaff showed that the molar ratios of ratios of adenine to thymine and adenine to thymine and guanine to cytosine are always guanine to cytosine are always approximately 1: 1 approximately 1: 1 in any DNA in any DNA structure although the ratio of structure although the ratio of adenine to guanine varies adenine to guanine varies according to the species from according to the species from which the DNA is obtained. which the DNA is obtained.

• This and other experimental This and other experimental observations lead observations lead Crick and Watson Crick and Watson in in 1953 to propose that the three-1953 to propose that the three-dimensional structure of DNA consisted dimensional structure of DNA consisted of two single molecule polymer chains of two single molecule polymer chains held together in the form of held together in the form of a double a double helix by hydrogen bonding between helix by hydrogen bonding between the same pairs of bases,the same pairs of bases, namely: namely:

prof. azaprof. aza

•the the adenineadenine-- thymine thymine and and cytosine-guanine base pairs cytosine-guanine base pairs (Figure 10.6). These pairs of (Figure 10.6). These pairs of bases, which are referred to bases, which are referred to as as complementary base pairscomplementary base pairs, form , form the internal structure of the the internal structure of the helix. helix.

prof. azaprof. aza

prof. azaprof. aza

•They are hydrogen bonded in They are hydrogen bonded in such a manner that their flat such a manner that their flat structures lie parallel to one structures lie parallel to one another across the inside of the another across the inside of the helix. The two polymer chains helix. The two polymer chains forming the helix are aligned in forming the helix are aligned in opposite directions. In other opposite directions. In other wordswords,, at the ends of the at the ends of the structure one chain has structure one chain has a free 3’-a free 3’-OH group and the other chain OH group and the other chain has a free 5’-OH group. has a free 5’-OH group.

• X-ray diffraction studies have since X-ray diffraction studies have since confirmed that this is the basic three confirmed that this is the basic three dimensional shape of the polymer dimensional shape of the polymer chains of the β -DNA, the natural chains of the β -DNA, the natural form of DNA. form of DNA.

• This form of DNA has about This form of DNA has about ten ten bases per turn of the helix. bases per turn of the helix.

prof. azaprof. aza

• Its outer surface has two grooves Its outer surface has two grooves known as the minor and major known as the minor and major grooves, respectively, which act as grooves, respectively, which act as the binding sites for many ligands. the binding sites for many ligands. Two other forms of DNA, the A and Z Two other forms of DNA, the A and Z forms, have also been identified but forms, have also been identified but it is not certain if these forms occur it is not certain if these forms occur naturally in living cells.naturally in living cells.

prof. azaprof. aza

prof. azaprof. aza

• Electron microscopy has shown that Electron microscopy has shown that the double helical chain of DNA is the double helical chain of DNA is folded, twisted and coiled into quite folded, twisted and coiled into quite compact shapes. A number of DNA compact shapes. A number of DNA structures are cyclic and these structures are cyclic and these compounds are also coiled and twisted compounds are also coiled and twisted into specific shapes. These shapes are into specific shapes. These shapes are referred to as supercoils, supertwists referred to as supercoils, supertwists and superhelices as appropriate.and superhelices as appropriate.

prof. azaprof. aza

prof. azaprof. aza

The two strands of DNA are held together by hydrogen bonds between bases. The sugars in the backbone are shown in light blue.

• Figure 10.6. The double helical Figure 10.6. The double helical structure of B-DNA. Interchanging of structure of B-DNA. Interchanging of either the bases of a base pair and/or either the bases of a base pair and/or base pair with base pair does not base pair with base pair does not affect the geometry of this structure. affect the geometry of this structure. Reproduced from G. Thomas. Reproduced from G. Thomas. (Chemistry (Chemistry for Pharmacy and the Life Sciences including Pharmacology for Pharmacy and the Life Sciences including Pharmacology and Biomedical Science, I996, by per mission of Prentice and Biomedical Science, I996, by per mission of Prentice Hall, a Pearson Education Company.Hall, a Pearson Education Company.

prof. azaprof. aza

prof. azaprof. aza

33.. The General Functions of The General Functions of DNADNA

The DNA found in the nuclei of cells The DNA found in the nuclei of cells has three functions:has three functions:

(i) to act as a repository for the genetic (i) to act as a repository for the genetic information required by a cell to information required by a cell to reproduce that cell:reproduce that cell:

(ii) to reproduce itself in order to (ii) to reproduce itself in order to maintain the genetic pool when cells maintain the genetic pool when cells divide;divide;

(ii(iiii) to supply the information that the ) to supply the information that the cell requires to manufacture specific cell requires to manufacture specific proteins.proteins.

• Genetic information is stored in a form Genetic information is stored in a form known as genes known as genes by the DNA found in by the DNA found in the nucleus of a cell (see section 10.4).the nucleus of a cell (see section 10.4).

• The duplication of DNA is known as The duplication of DNA is known as replicationreplication. It results in the formation of . It results in the formation of two identical DNA two identical DNA mmolecuoleculles that carry es that carry the same genetic information from the the same genetic information from the original cell original cell

prof. azaprof. aza

• to the two new cells that are formed to the two new cells that are formed when a cell divides (see section when a cell divides (see section 10.5).10.5).

• The function of DNA in protein The function of DNA in protein synthesis is to act synthesis is to act as a template for as a template for the production of the various RNA the production of the various RNA molecules necessary to produce a molecules necessary to produce a specific protein (see section 6)specific protein (see section 6)

prof. azaprof. aza

prof. azaprof. aza

Figure 10.7. A schematic representation of the gene for the ββ -subunit of haemoglobin.

prof. azaprof. aza

4. Genes4. Genes

• Each species has its own internal and Each species has its own internal and external characteristics. These external characteristics. These characteristics are determined by the characteristics are determined by the information stored and supplied by information stored and supplied by the DNA in the nuclei of its cells. the DNA in the nuclei of its cells.

• This information is carried This information is carried in the in the form of a code based on the form of a code based on the consecutive sequences of bases consecutive sequences of bases found in sections of the DNA found in sections of the DNA structurestructure (see section 5). (see section 5).

• This code controls the production of This code controls the production of the peptides and proteins required the peptides and proteins required by the body. by the body.

• The The sequence of bases that act as sequence of bases that act as the code for the production of one the code for the production of one specific peptide specific peptide or protein molecule or protein molecule is known as is known as a gene.a gene.

prof. azaprof. aza

prof. azaprof. aza

Changing the sequence of the bases effect Changing the sequence of the bases effect on the external or internal characteristics of on the external or internal characteristics of an individualan individual

• Genes can normally contain from Genes can normally contain from several hundred to 2000 bases. several hundred to 2000 bases. Changing the sequence of the basesChanging the sequence of the bases in a gene by in a gene by adding, subtracting or adding, subtracting or changing one or more baseschanging one or more bases may may cause a change in the structure of cause a change in the structure of that protein with a subsequent that protein with a subsequent knock-on knock-on effect on the external or effect on the external or internal characteristics of an internal characteristics of an individualindividual. .

• For example, an individual may have For example, an individual may have brown instead of blue eyes or their brown instead of blue eyes or their insulin production may be inhibited, insulin production may be inhibited, which could result in that individual which could result in that individual suffering from diabetes. suffering from diabetes.

• A number of medical conditions have A number of medical conditions have been attributed been attributed to either the absence to either the absence of a gene or the presence of a of a gene or the presence of a degenerate or faulty genedegenerate or faulty gene in which in which one or more of the bases in the one or more of the bases in the sequence have been changed.sequence have been changed.

prof. azaprof. aza

prof. azaprof. aza

• In simple organisms, such as bacteria, In simple organisms, such as bacteria, genetic information is usually stored in genetic information is usually stored in a continous sequence of DNA bases. a continous sequence of DNA bases.

• However, in higher organisms the However, in higher organisms the bases forming a particular gene may bases forming a particular gene may occur in a number of separate occur in a number of separate sections sections known as exonsknown as exons, separated , separated by sections of DNA that do not appear by sections of DNA that do not appear to be a code for any process. to be a code for any process.

•These non-coding sections are These non-coding sections are referred to referred to as intronsas introns. For . For example, the gene responsible for example, the gene responsible for the β-subunit of haemoglobin the β-subunit of haemoglobin consists of 990 bases. These bases consists of 990 bases. These bases occur occur as three exons separated by as three exons separated by two introns two introns (Figure 10.7).(Figure 10.7).

prof. azaprof. aza

prof. azaprof. aza

•The complete set of genes that The complete set of genes that contain all the hereditary contain all the hereditary information of a particular information of a particular species is called a genome. species is called a genome.

•The Human Genome Project. The Human Genome Project. initiated in 1990initiated in 1990, sets out to , sets out to identify all the genes that occur identify all the genes that occur in human chromosomes and also in human chromosomes and also the sequence of bases in these the sequence of bases in these genes. genes.

•This will create an index that can This will create an index that can be used to locate the genes be used to locate the genes responsible for particular medical responsible for particular medical conditions. For example, the gene conditions. For example, the gene in region q31 of chromosome 7 is in region q31 of chromosome 7 is responsible for the protein that responsible for the protein that controls the flow of chloride ions controls the flow of chloride ions through the membranes through the membranes in the in the lungs. lungs.

prof. azaprof. aza

•The changing of about three The changing of about three bases in exon number 10 gives a bases in exon number 10 gives a degenerate gene that is known degenerate gene that is known to to bbe responsible for causing e responsible for causing cystic fibrosis cystic fibrosis in a large number in a large number of cases.of cases.

prof. azaprof. aza

prof. azaprof. aza

Figure 10.8. A schematic representation of the replication of DNA. The arrows show the direction of growth of the leading and lagging strands. Reproduced from G. Thomas, Chemistry for Pharmacy and the

Life Sciences including Pharmacology and Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.

•Figure 10.8. A schematic Figure 10.8. A schematic representation of the replication of representation of the replication of DNA. The arrows show the direction DNA. The arrows show the direction of growth of the leading and of growth of the leading and lagging strands. lagging strands.

• Reproduced from G. Thomas, Chemistry for Pharmacy Reproduced from G. Thomas, Chemistry for Pharmacy and the Life Sciences including Pharmacology and and the Life Sciences including Pharmacology and Biomedical Science, 1996, by permission of Prentice Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.Hall, a Pearson Education Company.

prof. azaprof. aza

prof. azaprof. aza

DNA replication. The double helix (blue) is unwound by a helicase. Next, DNA polymerase III (green) produces the leading strand copy (red). A DNA polymerase I molecule (green) binds to the lagging strand. This enzyme makes discontinuous segments (called Okazaki fragments) before DNA ligase (violet) joins them together.

prof. azaprof. aza

5. Replication5. Replication

• Replication is believed to start with Replication is believed to start with the unwinding of a section of the the unwinding of a section of the double helix (Figure 10.8). double helix (Figure 10.8).

• Unwinding may start at the end or Unwinding may start at the end or more commonly in a central section more commonly in a central section of the DNA helix. of the DNA helix. It is initiated by the It is initiated by the binding of the DNA to specific binding of the DNA to specific receptor proteins receptor proteins that have been that have been activated by the appropriate first activated by the appropriate first messengemessenger r (see section 8.4). (see section 8.4).

• The separated strands of the DNA act The separated strands of the DNA act as as templates for the formation of a templates for the formation of a new daughter strandnew daughter strand. .

• Individual nucleotides, which are Individual nucleotides, which are synthesised in the cell by a complex synthesised in the cell by a complex route, bind by hydrogen bonding route, bind by hydrogen bonding between the bases to the between the bases to the complementary parent nucleotides. complementary parent nucleotides.

prof. azaprof. aza

•This hydrogen bonding is This hydrogen bonding is specific: specific: only the complementary only the complementary base pairs can hydrogen bond. base pairs can hydrogen bond.

• In other words, the hydrogen In other words, the hydrogen bonding can only bonding can only bbe between e between either either thymine and adenine thymine and adenine or or cytosine and guaninecytosine and guanine. .

prof. azaprof. aza

•This means that This means that the new the new daughter strand is an exact daughter strand is an exact replica of the original DNA strandreplica of the original DNA strand bound to the parent strand. bound to the parent strand.

•Consequently, replication will Consequently, replication will produce two identical DNA produce two identical DNA molecules.molecules.

prof. azaprof. aza

prof. azaprof. aza

• As the nucleotides hydrogen bond to As the nucleotides hydrogen bond to the parent strand they are linked to the parent strand they are linked to the adjacent nucleotide, which is the adjacent nucleotide, which is already hydrogen bonded to the already hydrogen bonded to the parent strand, by the action of parent strand, by the action of enzymes known as enzymes known as DNA polymerases. DNA polymerases.

• As the daughter strands grow, the As the daughter strands grow, the DNA helix continues to unwind. DNA helix continues to unwind.

• However, both daughter strands are However, both daughter strands are formed at the same time formed at the same time in the 5’ to in the 5’ to the 3’ direction. the 3’ direction.

• This means that the growth of the This means that the growth of the daughter strand that starts at the 3’ daughter strand that starts at the 3’ end of the parent strand can continue end of the parent strand can continue smoothly as the DNA helix continues smoothly as the DNA helix continues to unwind. to unwind.

• This strand is known the leading This strand is known the leading strand. However, this smooth growth strand. However, this smooth growth is riot possible for the daughter strand is riot possible for the daughter strand that started from the 5’ of the parent that started from the 5’ of the parent strand. strand.

prof. azaprof. aza

• This strand, known This strand, known as the lagging as the lagging strand,strand, is formed in a series of is formed in a series of sections, each of which still grows in sections, each of which still grows in the 5’ to 3’ direction. the 5’ to 3’ direction.

• These sections. which are known as These sections. which are known as Okazaki fragments after their Okazaki fragments after their discoverer, are joined together by discoverer, are joined together by the the enzyme DNA ligaseenzyme DNA ligase to form the to form the second daughter strand.second daughter strand.

prof. azaprof. aza

prof. azaprof. aza

• ReplicationReplication, , which starts at the end which starts at the end of a DNA helix, continues of a DNA helix, continues uuntil the ntil the entire structure has been duplicated. entire structure has been duplicated.

• The same result is obtained when The same result is obtained when replication starts at the centre of a replication starts at the centre of a DNA helix. DNA helix.

• In this case, In this case, unwinding continues in unwinding continues in both directions until the complete both directions until the complete molecule is duplicated.molecule is duplicated. This latter This latter situation is more common.situation is more common.

• DNA replication occurs when cell DNA replication occurs when cell division is division is imminentimminent. At the same time, . At the same time, new histones are synthesised. new histones are synthesised.

• This results in a thickening of the This results in a thickening of the chromatin filaments into chromosomes chromatin filaments into chromosomes (see section 2). These rod-like (see section 2). These rod-like structures can be stained and are large structures can be stained and are large enough to be seen under a microscope.enough to be seen under a microscope.

prof. azaprof. aza

prof. azaprof. aza

6. Ribonucleic Acids (RNA)6. Ribonucleic Acids (RNA)

• Ribonucleic acids are found in both Ribonucleic acids are found in both the the nucleus and the cytoplasmnucleus and the cytoplasm. In . In the cytoplasm RNA is located mainly the cytoplasm RNA is located mainly in small spherical organelles known in small spherical organelles known as as ribosome.ribosome. These consist of about These consist of about 65% RNA and 35% protein. 65% RNA and 35% protein.

• Ribonucleic acids are classified Ribonucleic acids are classified according to their general role in according to their general role in protein synthesis as: messenger RNA protein synthesis as: messenger RNA (mRNA): transfer RNA (tRNA): and (mRNA): transfer RNA (tRNA): and ribosomal RNA (rRNA). ribosomal RNA (rRNA).

• Messenger Messenger RNA informs the ribosome as RNA informs the ribosome as to what amino acids are required to what amino acids are required and and their order in the protein, that is, they their order in the protein, that is, they carry the genetic information necessary carry the genetic information necessary to produce a specific protein. to produce a specific protein.

• This type of RNA is synthesised as This type of RNA is synthesised as required and once its message has been required and once its message has been delivered it is decomposed. delivered it is decomposed.

prof. azaprof. aza

prof. azaprof. aza

Figure 10.9. (a) The general structure ol a section of an RNA polymer chain. (b) The hydrogen bonding between uracil and adenine. Reproduced from G.’Thomas, Chemistry to Pharmacy and the Life Science including Pharmacology including Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.

• Figure 10.9. (a) The general structure Figure 10.9. (a) The general structure ooff a section of an RNA polymer chain. a section of an RNA polymer chain. (b) The hydrogen bonding between (b) The hydrogen bonding between uracil and adenine. uracil and adenine. Reproduced from Reproduced from G.’Thomas, Chemistry to Pharmacy and the Life Science G.’Thomas, Chemistry to Pharmacy and the Life Science including Pharmacology including Biomedical Science, including Pharmacology including Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education 1996, by permission of Prentice Hall, a Pearson Education Company. Company.

prof. azaprof. aza

prof. azaprof. aza

Figure 10.10. A schematic representation of a transcription process. Reproduced from G.’Thomas, Chemistry to Pharmacy and the Life Science including Pharmacology including Biomedical Science, 1996, by permission of Prentice Hall, a Pearson Education Company.

prof. azaprof. aza

• The structures of RNA molecules The structures of RNA molecules consist of a single polymer chain of consist of a single polymer chain of nucleotides with the same bases as nucleotides with the same bases as DNA, with the DNA, with the exception of thymine, exception of thymine, which is replaced by uracilwhich is replaced by uracil ( Figure ( Figure 9).9).

• These chains often contain single-These chains often contain single-stranded loops separated by short stranded loops separated by short sections of a distorted double helix sections of a distorted double helix (Figure 11). These structures are (Figure 11). These structures are known as hairpin loops. known as hairpin loops.

• All types of RNA are formed from DNA All types of RNA are formed from DNA by a process known by a process known as transcriptionas transcription. It . It is thought that the DNA unwinds and is thought that the DNA unwinds and the RNA molecule is formed in the 5’ to the RNA molecule is formed in the 5’ to 3’ direction. 3’ direction.

• It proceeds smoothly. with the 3’ end of It proceeds smoothly. with the 3’ end of the new strand bonding to the 5’ end of the new strand bonding to the 5’ end of the next nucleotide (Figure 10.10). the next nucleotide (Figure 10.10).

prof. azaprof. aza

prof. azaprof. aza

• This bonding is catalysed by enzymes This bonding is catalysed by enzymes known as known as RNA polymerasesRNA polymerases. .

• The sequence of bases in the new RNA The sequence of bases in the new RNA strand is controlled by the sequence of strand is controlled by the sequence of bases in the parent DNA strand. bases in the parent DNA strand.

• In this way In this way DNA controls the genetic DNA controls the genetic information being transcribed into the information being transcribed into the RNA molecule. RNA molecule.

• The strands of DNA also contain start The strands of DNA also contain start and stop signals, which control the size and stop signals, which control the size of the RNA molecule produced. of the RNA molecule produced.

• These signals are in the form of specific These signals are in the form of specific sequences of bases. sequences of bases.

• It is believed that the enzyme It is believed that the enzyme rhorho factor factor could be involved in the termination of could be involved in the termination of the synthesis and the release of some the synthesis and the release of some RNA molecules from the parent DNA RNA molecules from the parent DNA strand. However, in many cases there is strand. However, in many cases there is no evidence that this enzyme is involved no evidence that this enzyme is involved in the release of the RNA molecule.in the release of the RNA molecule.

prof. azaprof. aza

•The RNA produced within the The RNA produced within the nucleus by transcription is known nucleus by transcription is known as heterogeneousas heterogeneous nuclear RNA nuclear RNA (hnRNA), premessenger RNA (hnRNA), premessenger RNA (pre-mRNA) or primary transcript (pre-mRNA) or primary transcript RNARNA ( ptRNA). ( ptRNA).

prof. azaprof. aza

• Since the DNA gene from which it is Since the DNA gene from which it is produced contains both exons and produced contains both exons and introns, the hnRNA will also contain introns, the hnRNA will also contain its genetic information in the form of its genetic information in the form of a series of exons and introns a series of exons and introns complementary to those of its parent complementary to those of its parent gene.gene.

prof. azaprof. aza

prof. azaprof. aza

7. Messenger RNA (mRNA)7. Messenger RNA (mRNA)

• mmRNA RNA carries the genetic message from carries the genetic message from the DNA in the nucleus to a ribosomethe DNA in the nucleus to a ribosome. . This message instructs the ribosome to This message instructs the ribosome to synthesise a specific protein. synthesise a specific protein.

• mRNA is believed to be produced in the mRNA is believed to be produced in the nucnuclleus from hnRNA eus from hnRNA by removal of the by removal of the introns and the splicing together ointrons and the splicing together off the the remaining exons into a continuous remaining exons into a continuous genetic message, genetic message,

• the process being catalysed by the process being catalysed by specialised enzymes. The net result specialised enzymes. The net result is a smaller mRNA molecule with a is a smaller mRNA molecule with a continuous sequence of bases continuous sequence of bases that that are complementary to the gene’s are complementary to the gene’s exonsexons, this mRNA now leaves the , this mRNA now leaves the nucleus and carries its message in nucleus and carries its message in the form of a code to a ribosome.the form of a code to a ribosome.

prof. azaprof. aza

prof. azaprof. aza

prof. azaprof. aza

all protein synthesis starts with all protein synthesis starts with methioninemethionine• The code carried by mRNA was The code carried by mRNA was

broken in the 1960s by Nirenberg broken in the 1960s by Nirenberg and other workers. These workers and other workers. These workers demonstrated that demonstrated that each naturally each naturally occurring amino acid had a DNA code occurring amino acid had a DNA code that consisted of a sequence of three that consisted of a sequence of three consecutive bases known as a codonconsecutive bases known as a codon and that an amino acid could have and that an amino acid could have several different codons (Table 10.1), several different codons (Table 10.1),

• In addition, three of the codons are In addition, three of the codons are stop signals which instruct the stop signals which instruct the ribosome to sribosome to sttop protein synthesis. op protein synthesis.

• Furthermore, the codon Furthermore, the codon that initiates that initiates the synthesis is always AUG, which is the synthesis is always AUG, which is also the codon for methioninealso the codon for methionine. . Consequently, all protein synthesis Consequently, all protein synthesis starts with methionine. starts with methionine. However, few completed proteins However, few completed proteins have a terminal methionine because have a terminal methionine because this residue is normally removedthis residue is normally removed before the peptide chain is complete. before the peptide chain is complete.

prof. azaprof. aza

•Moreover, methionine can still Moreover, methionine can still bbe e incorporated in a peptide chain incorporated in a peptide chain because there are two different because there are two different tRNAs that transfer methionine tRNAs that transfer methionine to the ribosome (see section 8). to the ribosome (see section 8).

prof. azaprof. aza

prof. azaprof. aza

all living matter using the same genetic all living matter using the same genetic code for protein synthesiscode for protein synthesis

•One is specific for the transfer of One is specific for the transfer of the initial methionine whereas the the initial methionine whereas the other will only deliver methionine other will only deliver methionine to the developing peptide chain, to the developing peptide chain, By convention, the three letters By convention, the three letters of codon triplets are normally of codon triplets are normally written with their 5’ ends on the written with their 5’ ends on the left and their 3’ ends on the rightleft and their 3’ ends on the right..

•The mRNA’s codon code is known The mRNA’s codon code is known as the genetic code, Its use is as the genetic code, Its use is universal, universal, all living matter using all living matter using the same genetic code for protein the same genetic code for protein synthesis. synthesis.

•This suggests that all living matter This suggests that all living matter must have originated from the must have originated from the same source and is strong same source and is strong evidence for Darwin’s theory of evidence for Darwin’s theory of evolution.evolution.

prof. azaprof. aza

• Figure .11. The general structures of Figure .11. The general structures of tRNA. (a) The two-dimensional tRNA. (a) The two-dimensional cloverleaf representation showing some cloverleaf representation showing some of the invariable nucleotides that occur of the invariable nucleotides that occur in the same positions in most tRNA in the same positions in most tRNA molecules and (molecules and (bb) the three- ) the three- dimensional L shape dimensional L shape (From CHEMISTRY, by (From CHEMISTRY, by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and Company)of W. H. Freeman and Company)

prof. azaprof. aza

prof. azaprof. aza

8. Transfer RNA (tRNA)8. Transfer RNA (tRNA)

• tRNAs are also believed to be formed tRNAs are also believed to be formed in the nucleus from the hnRNA. in the nucleus from the hnRNA.

• They are relatively small molecules They are relatively small molecules that usually that usually contain from 73 to 94 contain from 73 to 94 nucleotides in a single strand. Some nucleotides in a single strand. Some of these nucleotides may contain of these nucleotides may contain derivatives of the principal bases, derivatives of the principal bases, such as such as 2’-O-methylguanosine (0MG) 2’-O-methylguanosine (0MG) and inosine (I).and inosine (I).

• The strand of tRNA is usually folded The strand of tRNA is usually folded into a into a three-dimensional L shapethree-dimensional L shape. .

•This structure, which consists of This structure, which consists of several loops, is several loops, is held in this shape held in this shape by hydrogen bondingby hydrogen bonding between between complementary base pairs in the complementary base pairs in the stem sections of these loops and stem sections of these loops and also by hydrogen bonding also by hydrogen bonding between bases in different loops. between bases in different loops.

prof. azaprof. aza

prof. azaprof. aza

Figure .11. The general structures of tRNA. (a) The two-dimensional cloverleaf representation showing some of the invariable nucleotides that occur in the same positions in most tRNA molecules and (b) the three- dimensional L shape (From CHEMISTRY, by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and Company)

• Figure .11. The general structures of Figure .11. The general structures of tRNA. (a) The two-dimensional tRNA. (a) The two-dimensional cloverleaf representation showing cloverleaf representation showing some of the invariable nucleotides some of the invariable nucleotides that occur in the same positions in that occur in the same positions in most tRNA molecules and (most tRNA molecules and (bb) the ) the three- dimensional L shape three- dimensional L shape (From (From CHEMISTRY, by Linus Pauling and Peter Pauling. CHEMISTRY, by Linus Pauling and Peter Pauling. Copyright © 1975 by Linus Pauling and Peter Copyright © 1975 by Linus Pauling and Peter Paling. Used with permission of W. H. Freeman and Paling. Used with permission of W. H. Freeman and Company)Company)

prof. azaprof. aza

•This results in the formation of This results in the formation of sections of double helical sections of double helical sstructures. tructures.

•However, the structures of most However, the structures of most tRNAs are represented in two tRNAs are represented in two dimensions dimensions as a cloverleaf as a cloverleaf (Figure (Figure 11).11).

prof. azaprof. aza

prof. azaprof. aza

•tRNA molecules tRNA molecules carry amino acid carry amino acid residues from the cell’s amino residues from the cell’s amino acid pool to the mRNA attached acid pool to the mRNA attached to the ribosome. to the ribosome.

•The amino acid residue is The amino acid residue is attached through an ester attached through an ester linkage to ribosome residue linkage to ribosome residue at at the 3’ terminal of the tRNA the 3’ terminal of the tRNA strandstrand, which almost invariably , which almost invariably has the sequence CCA. has the sequence CCA.

• This sequence plus a fourth nucleotide This sequence plus a fourth nucleotide projects beyond the double helix of the projects beyond the double helix of the stem. Each type of amino acid can only stem. Each type of amino acid can only be transported be transported byby its own specific tRNA its own specific tRNA molecule. molecule. In other words a tRNA that In other words a tRNA that carries serine residues will not carries serine residues will not transport alanine residuestransport alanine residues. In other . In other word, some amino acids can word, some amino acids can bbe carried e carried by several different tRNA moleculesby several different tRNA molecules

prof. azaprof. aza

prof. azaprof. aza

• The tRNA recognises the point on the The tRNA recognises the point on the mRNA where it has to deliver its mRNA where it has to deliver its amino acid through the use of a amino acid through the use of a group of three bases known group of three bases known as an as an anticodon. anticodon.

• This anticodon is a sequence of three This anticodon is a sequence of three bases found on one of the loops of bases found on one of the loops of the tRNA (Figure 11). the tRNA (Figure 11).

• The The anticodon can only form base anticodon can only form base with the complementary codon in the with the complementary codon in the mRNAmRNA. .

prof. azaprof. aza

•Consequently, the tRNA Consequently, the tRNA wwill only ill only hydrogen bond to the region of hydrogen bond to the region of the mRNA that has the correct the mRNA that has the correct codon, which means codon, which means its amino its amino acid can only be delivered to a acid can only be delivered to a specific point on the mRNA. specific point on the mRNA.

prof. azaprof. aza

• For example, a tRNA molecule with For example, a tRNA molecule with the the anantticodon CGA will only transport icodon CGA will only transport its alanine residue to a GCU codon its alanine residue to a GCU codon on on the mRNA.the mRNA.

• Furthermore, this mechanism will Furthermore, this mechanism will also control also control the order in which amino the order in which amino acid residues are added to the acid residues are added to the growing protein.growing protein.

prof. azaprof. aza

prof. azaprof. aza

99.. Ribosomal RNA (rRNA) Ribosomal RNA (rRNA)

• Ribosomes contain about 35% protein Ribosomes contain about 35% protein and 65% rRNA. and 65% rRNA.

• Their structures are complex and Their structures are complex and have not yet been fully elucidated. have not yet been fully elucidated.

• However, they have been found to However, they have been found to consist of two Sections that are consist of two Sections that are referred to referred to as the large and small as the large and small subunits. subunits.

• Each of these subunits contains Each of these subunits contains protein and rRNAprotein and rRNA

• In Eschericia coli the small subunit In Eschericia coli the small subunit has been shown to contain a 1542-has been shown to contain a 1542-nucleotide rRNA molecule whereas nucleotide rRNA molecule whereas the large contains two rRNA the large contains two rRNA molecules of 120 (Figure 12) and molecules of 120 (Figure 12) and 2094 nucleotides, respectively.2094 nucleotides, respectively.

prof. azaprof. aza

prof. azaprof. aza

• Experimental evidence suggests that Experimental evidence suggests that rRNA molecules have structures that rRNA molecules have structures that consist of consist of a single strand of a single strand of nucleotides whose sequence varies nucleotides whose sequence varies considerably from species to species. considerably from species to species.

• The strand is folded and twisted to The strand is folded and twisted to form a series of single-stranded loops form a series of single-stranded loops separated by sections of double helix separated by sections of double helix (Figure 12). (Figure 12).

prof. azaprof. aza

Figure 10.12. The proposed sequence of nucleotides in the 120-nucleotide subunit found in Escherichia coli ribosome showing the single-stranded loops and the double helical structures. (Reprinted, with permission, from the Annual Review of Biochemistry, volume 53 © I984 by Annual Reviews. www.Annual Reviews.org).

• The double helical segments are The double helical segments are believed to be formed by hydrogen believed to be formed by hydrogen bonding between complementary bonding between complementary base pairs. base pairs.

• The The general pattern of loops and general pattern of loops and helixes is very similar between helixes is very similar between species even though the sequence of species even though the sequence of nucleotides are differentnucleotides are different

prof. azaprof. aza

•However, little is known about However, little is known about the three-dimensional structures the three-dimensional structures of rRNA molecules of rRNA molecules and their and their interactions with the proteins interactions with the proteins found in the rifound in the ribbosome.osome.

prof. azaprof. aza

prof. azaprof. aza

10. Protein Synthesis10. Protein Synthesis

•Protein synthesis starts from the Protein synthesis starts from the N-terminal of the protein. N-terminal of the protein.

• IIt t proceeds in the 5’ to 3’ proceeds in the 5’ to 3’ direction along the mRNAdirection along the mRNA and and may be divided into four mayor may be divided into four mayor stages. namely: stages. namely: activation: activation: initiation: elongation: and initiation: elongation: and termination. termination.

ActivationActivation

•ActivationActivation is the formation of the is the formation of the ttRNA amino acid complex. RNA amino acid complex.

• InitiationInitiation is the binding of the is the binding of the mRNA to the ribosome and the mRNA to the ribosome and the activation of the ribosome. activation of the ribosome. ElongationElongation is the formation of the is the formation of the protein. protein.

prof. azaprof. aza

• TerminationTermination is the ending of the protein is the ending of the protein synthesis and its release from the synthesis and its release from the ribosome. ribosome.

• All these processes normally require All these processes normally require the participation of the participation of protein catalysts, protein catalysts, known as factors,known as factors, as well as other as well as other proteins whose function is not always proteins whose function is not always known. known.

• GTP and sometimes ATP act as sources GTP and sometimes ATP act as sources of energy of energy for the processes.for the processes.

prof. azaprof. aza

prof. azaprof. aza

10.1 Activation10.1 ActivationIt is believed that the amino acids from the cellular pool react with ATP to form an active amino acid-AMP complex. This complex reacts with the specific tRNA for the amino acid. the reaction being catalysed by a synthese that is specific for that amino acid.

• It is believed that the amino acidsIt is believed that the amino acids ( ( AAA) A) from the cellular pool react with from the cellular pool react with ATP to form ATP to form an active amino acid-AMP an active amino acid-AMP complexcomplex (AA-AMP) (AA-AMP). .

• This complex reacts with This complex reacts with the specific the specific tRNA for the amino acidtRNA for the amino acid, , the reaction the reaction being catalysed by being catalysed by a syntha synthaase se that is that is specific for that amino acid.specific for that amino acid.

prof. azaprof. aza

prof. azaprof. aza

Figure 13. A schematic representation of the initiation of protein synthesis.

prof. azaprof. aza

22.. Initiation Initiation• The general mechanism of initiation The general mechanism of initiation

is well documented but the liner is well documented but the liner details are still not known. details are still not known.

• It is thought that it starts with the It is thought that it starts with the two subunits of the ribosome two subunits of the ribosome separating and the binding of the separating and the binding of the mRNA to the smaller subunit. mRNA to the smaller subunit.

• Protein synthesis then starts by the Protein synthesis then starts by the attachment of a methionine-tRNA attachment of a methionine-tRNA complex to the mRNA complex to the mRNA so that it forms so that it forms the N-terminal of the new protein. the N-terminal of the new protein.

• Methionine is always the first amino Methionine is always the first amino acid in all protein synthesis because acid in all protein synthesis because its tRNA anticodon is also the signal its tRNA anticodon is also the signal for the ribosome system to start for the ribosome system to start protein synthesis. protein synthesis.

• Because Because the anticodon for the anticodon for methionine tRNA is UACmethionine tRNA is UAC, this , this synthesis will start synthesis will start at the AUG codon at the AUG codon of the mRNAof the mRNA. .

prof. azaprof. aza

•This codon is usually found This codon is usually found within the first 30 nucleotides of within the first 30 nucleotides of the mRNA. the mRNA.

•However, few proteins have an However, few proteins have an N-terminal methionine because N-terminal methionine because once protein synthesis has once protein synthesis has started the methionine started the methionine is usually is usually removed by hydrolysis. removed by hydrolysis.

prof. azaprof. aza

prof. azaprof. aza

• As soon as As soon as the methionine-tRNA has the methionine-tRNA has bound to the mRNAbound to the mRNA the larger the larger ribosomal subunit is believed to bind ribosomal subunit is believed to bind to the smaller subunit to the smaller subunit so that the so that the mRNA is sandwiched mRNA is sandwiched between the between the two subunits (Figure 13). two subunits (Figure 13).

• This large subunit is believed to have This large subunit is believed to have three binding sites called the three binding sites called the P P (peptidyl), A (acceptor) and E (exit) (peptidyl), A (acceptor) and E (exit) sites.sites.

• It attaches itself to the smaller It attaches itself to the smaller subunit so subunit so that its P site is aligned that its P site is aligned with the methionine- tRNA with the methionine- tRNA complex bound to the mRNAcomplex bound to the mRNA. .

•This P site is where the growing This P site is where the growing protein will be bound to the protein will be bound to the ribosomeribosome. .

prof. azaprof. aza

• The A site, which is thought to be The A site, which is thought to be adjacent to the P site, adjacent to the P site, is where the is where the next amino acid-next amino acid-ttRNA complex RNA complex bbinds inds to the ribosome so that its amino acid to the ribosome so that its amino acid can can bbe attached to the peptide chain. e attached to the peptide chain.

• The E site is where the discharged The E site is where the discharged tRNA is transiently bound before it tRNA is transiently bound before it leaves the ribosome.leaves the ribosome.

prof. azaprof. aza

prof. azaprof. aza

•This large subunit is believed to This large subunit is believed to have have three binding sites called three binding sites called the P (peptidthe P (peptidyyl), A (acceptor) and l), A (acceptor) and E (exit) sites. E (exit) sites.

• It attaches itself to the smaller It attaches itself to the smaller subunit so that its P site is subunit so that its P site is aligned with the methionine-tRNA aligned with the methionine-tRNA complex bound to the mRNA. complex bound to the mRNA.

•This P site is This P site is where the where the growing protein will be bound growing protein will be bound to the ribosometo the ribosome. .

prof. azaprof. aza

• The A site, which is thought to be The A site, which is thought to be adjacent to the P site, adjacent to the P site, is where the is where the next amino acid-tRNA complex binds next amino acid-tRNA complex binds to the ribosome so that its amino acid to the ribosome so that its amino acid can be attached to the peptide chaincan be attached to the peptide chain. .

• The E site is where The E site is where the discharged the discharged tRNA is transiently tRNA is transiently bbound before it ound before it leaves the ribosomeleaves the ribosome

prof. azaprof. aza

prof. azaprof. aza

33.. Elongation Elongation•Elongation is the formation of Elongation is the formation of

the peptide chain of the the peptide chain of the protein protein by a stepwise by a stepwise repetitive process.repetitive process.

•A great deal is known about A great deal is known about the nature of this process the nature of this process bbut ut its exact mechanism is still not its exact mechanism is still not fully understood. fully understood.

•The process of elongation is best The process of elongation is best explained by the use of a explained by the use of a hypothetical example. hypothetical example.

•Suppose that the sequence of Suppose that the sequence of codons, including the start codons, including the start codon, is codon, is AUGAUGUUGUUGGCUGCUGGAGGA.. etc.. etc

prof. azaprof. aza

•The elongation process starts The elongation process starts with with the methionine-tRNA bound the methionine-tRNA bound to the AUG codon of the mRNA to the AUG codon of the mRNA (Figure 14). (Figure 14).

•Because the second codon is Because the second codon is UUG UUG the second amino acid in the second amino acid in the polypetide chain will be the polypetide chain will be leucine. leucine.

prof. azaprof. aza

•This amino acid is transported This amino acid is transported by by a tRNA molecule with the a tRNA molecule with the anticodon AAC anticodon AAC because this is the because this is the only anticodon that matches the only anticodon that matches the UUG codon on the mRNA strand. UUG codon on the mRNA strand.

•The leucine- tRNA complex ‘docks’ The leucine- tRNA complex ‘docks’ on the UUG codon of the mRNA on the UUG codon of the mRNA and and binds to the A site. binds to the A site.

prof. azaprof. aza

prof. azaprof. aza

• This docking and binding is believed This docking and binding is believed to involve rito involve ribbosome proteins, osome proteins, referred to as elongation factors, and referred to as elongation factors, and energy supplied by the hydrolysis of energy supplied by the hydrolysis of guanosine triphosphate (GTP) to guanosine triphosphate (GTP) to guanosine diphosphate (GDP). guanosine diphosphate (GDP).

• Once the leucine-tRNA has occupied Once the leucine-tRNA has occupied the A site the methionin is linked to the A site the methionin is linked to the leucine by means of a peptide the leucine by means of a peptide link link whose carbonyl group originates whose carbonyl group originates from the methionine. from the methionine.

• TThis rhis reeaction is catalysed by the action is catalysed by the appropriate transferase. appropriate transferase.

• It leaves the tRNA on the P site It leaves the tRNA on the P site empty and produces an empty and produces an (NH2)-(NH2)-Met-Leu-tRNA complex Met-Leu-tRNA complex at the A at the A sitesite. .

prof. azaprof. aza

•The empty tRNA is discharged The empty tRNA is discharged through the through the EE site and at the same site and at the same time time the complete ribosome the complete ribosome moves along the mRNA in the 5’ to moves along the mRNA in the 5’ to 3’ 3’ direction so that the direction so that the dipeptide-dipeptide-tRNA complex moves from the A tRNA complex moves from the A site to the P sitesite to the P site. .

prof. azaprof. aza

•This process is known as This process is known as translocationtranslocation. .

• It is poorly understood but It is poorly understood but it leaves it leaves the A site empty and able to the A site empty and able to receive the next amino acid tRNA receive the next amino acid tRNA complex.complex.

•The whole process is then repeated The whole process is then repeated in order to add the next amino acid in order to add the next amino acid residue to peptide chain. residue to peptide chain.

prof. azaprof. aza

• Because the next mRNA codon in our Because the next mRNA codon in our hypothetical example is (hypothetical example is (GCUGCU) this ) this amino acid will be amino acid will be alaninealanine (see Table (see Table 10. l). Subsequent amino acids are 10. l). Subsequent amino acids are added in a similar way, the sequence added in a similar way, the sequence of amino acid residues in tof amino acid residues in thehe chain chain being control led by the order of the being control led by the order of the codons in the mRNA.codons in the mRNA.

prof. azaprof. aza

• It is poorly understood but it It is poorly understood but it leaves the A site empty and able leaves the A site empty and able to receive the next amino acid to receive the next amino acid tRNA complex. The whole tRNA complex. The whole process is then repeated in order process is then repeated in order to add the next amino acid to add the next amino acid residue to peptide chain . residue to peptide chain .

prof. azaprof. aza

• Figure 13Figure 13.. A diagrammatic representation of A diagrammatic representation of the process of elongation in protein synthesisthe process of elongation in protein synthesis

prof. azaprof. aza

prof. azaprof. aza

10.4 10.4 TerminationTermination

•The elongation process continues The elongation process continues until a stop codon is reacheduntil a stop codon is reached..

• ThisThis codon cannot accept an codon cannot accept an amino acid-tRNA complex and so amino acid-tRNA complex and so the synthesis sthe synthesis sttops. ops.

•At this point the peptide-tRN At this point the peptide-tRN chain occupies a P site and chain occupies a P site and the A the A site is empty. site is empty.

•The stop codon of the mRNA is The stop codon of the mRNA is recognised by proteins know as recognised by proteins know as release factorsrelease factors, which promote , which promote the release of the protein from the release of the protein from the ribosome. the ribosome.

prof. azaprof. aza

•The mechanism by which this The mechanism by which this happens is not fully understood happens is not fully understood but they are believed convert the but they are believed convert the transferase responsible for transferase responsible for peptide synthesis into a peptide synthesis into a hydrolase, hydrolase, which catalyses which catalyses hydrolysis of the ester group hydrolysis of the ester group linking the polypeptide to its linking the polypeptide to its tRNA. tRNA.

prof. azaprof. aza

•Once released, the protein is Once released, the protein is folded into its characteristic folded into its characteristic shapeshape. often under the . often under the direction of molecular direction of molecular chaperone protein.chaperone protein.

prof. azaprof. aza

prof. azaprof. aza

11 Protein Synthesis in Prokaryotic and 11 Protein Synthesis in Prokaryotic and Eukaryotic CellsEukaryotic Cells

• The general sequence of events The general sequence of events protein synthesis is similar for both protein synthesis is similar for both eukaryotic and pro prokaryotic cells. eukaryotic and pro prokaryotic cells.

• In both cases In both cases the hydrolysis of GDP the hydrolysis of GDP to GDP is the source of energy for to GDP is the source of energy for many of the processes involvedmany of the processes involved..

• However, the structures of However, the structures of prokaryotic and eukaryotic prokaryotic and eukaryotic ribosomes are differentribosomes are different

•For example, the ribosomes of For example, the ribosomes of prokaryotic cells of bacteria are prokaryotic cells of bacteria are made up made up of 50Sof 50S (see Apendix 3) (see Apendix 3) and 30S rRNA and 30S rRNA subunits whereas subunits whereas the ribosthe ribosoomes of mammalian mes of mammalian eukaryotic cells consist ,of eukaryotic cells consist ,of 60S 60S and 40S rRNA and 40S rRNA subunits. subunits.

prof. azaprof. aza

•The differences between the The differences between the ribosomes of prokaryotic and ribosomes of prokaryotic and eukaryotic ribosomes eukaryotic ribosomes are the are the basis of the selective action of basis of the selective action of some antibioticssome antibiotics . .

prof. azaprof. aza

prof. azaprof. aza

11.111.1.. Prokaryotic CeLLs Prokaryotic CeLLs•The first step in protein synthesis The first step in protein synthesis

is the correct alignment of mRNA is the correct alignment of mRNA on the small subunit of the on the small subunit of the ribosome. ribosome.

• In prokaryotic cells this alignment In prokaryotic cells this alignment is believed to be due to binding is believed to be due to binding by base pairing by base pairing between bases at between bases at the 3’ end of the rRNA of the the 3’ end of the rRNA of the ribosome and bases at the 5’ end ribosome and bases at the 5’ end of the mRNAof the mRNA..

• This ensures the correct alignment of This ensures the correct alignment of the the AUG anticodon of the mRNA with AUG anticodon of the mRNA with the P site of the ribosomethe P site of the ribosome. .

• The mRNA sequence of bases The mRNA sequence of bases responsible for this binding occurs as responsible for this binding occurs as part of the upstream (5’ terminal part of the upstream (5’ terminal end) section of the strand before the end) section of the strand before the start codon. start codon.

prof. azaprof. aza

prof. azaprof. aza

• This sequence is often known as the This sequence is often known as the Shine-Dalgarno sequenceShine-Dalgarno sequence after its after its discovers. Shine-Dalgarno sequences discovers. Shine-Dalgarno sequences vary in length and base sequence vary in length and base sequence (Figure 10.15).(Figure 10.15).

• The initiating tRNA in prokaryotic The initiating tRNA in prokaryotic cells is a specific methionine-tRNA cells is a specific methionine-tRNA known as known as tRNAtRNAff

MetMet ,which is able to ,which is able to read the start codon AUG but not read the start codon AUG but not when it is part of the elongation when it is part of the elongation sequence. tRNAsequence. tRNAff

MetMet is unique in that is unique in that the methionine it carries is usually in the methionine it carries is usually in the form of its N-formyl derivative.the form of its N-formyl derivative.

prof. azaprof. aza

Figure 10.15. Examples of Shine-Dalgarno sequences (bold larger type) of mRNA recognised by Escherichia coli ribosomes. These sequences lie about 10 nucleotides upstream of the AUG start codon for the specified protein.

prof. azaprof. aza

When AUG is part of the elongation sequence methionine is added to the growing protein by a different transfer RNA known as tRNAtRNAmm

MetMet, which also has the anticodon UAC.

• However, tRNAmMet cannot initiate However, tRNAmMet cannot initiate protein synthesis. Elongation follows protein synthesis. Elongation follows the general mechanism for protein the general mechanism for protein synthesis (see section 10.9). synthesis (see section 10.9).

• It requires a group of proteins known It requires a group of proteins known as elongation factors and energy as elongation factors and energy supplied by the hydrolysis of GTP to supplied by the hydrolysis of GTP to GDP. Termination normally involves GDP. Termination normally involves three release factors.three release factors.

prof. azaprof. aza

prof. azaprof. aza

•Experimental work has shown Experimental work has shown that an mRNA strand actively that an mRNA strand actively ssyynthesinthesizzing proteins still have ing proteins still have several ribosomes attached to it several ribosomes attached to it at different places along its at different places along its length. These multiple ribosome length. These multiple ribosome structures are referred to as structures are referred to as polyribosomes or polysomes. polyribosomes or polysomes.

•The polysomes of prokaryotic The polysomes of prokaryotic cells can contain up to 10 cells can contain up to 10 ribosomes at any one lime. Each ribosomes at any one lime. Each of these ribosomes will be of these ribosomes will be simultaneously producing the simultaneously producing the same polypeptide or protein; same polypeptide or protein;

prof. azaprof. aza

•the further the ribosome has the further the ribosome has moved along the mRNA, the moved along the mRNA, the longer the polypeptide chain. The longer the polypeptide chain. The process resembles the assembly process resembles the assembly line in a factory. Each mRNA line in a factory. Each mRNA strand can in its lifetime produce strand can in its lifetime produce up to 300 protein molecules.up to 300 protein molecules.

prof. azaprof. aza

prof. azaprof. aza

10 amino acid residues are 10 amino acid residues are added..added..

• In prokaryotic but not eukaryotic In prokaryotic but not eukaryotic cells (see section 4.1), ribosomes are cells (see section 4.1), ribosomes are found in association with DNA. found in association with DNA.

• This is believed to he due to the This is believed to he due to the ribosome binding to the mRNA as it ribosome binding to the mRNA as it is produced by transcription from the is produced by transcription from the DNA. DNA.

•Furthermore, these ribosomes Furthermore, these ribosomes have been shown to start have been shown to start producing the polypeptide chain producing the polypeptide chain of their designated protein of their designated protein before transcription is complete. before transcription is complete.

prof. azaprof. aza

•This means that in bacteria protein This means that in bacteria protein synthesis can be very rapid and in synthesis can be very rapid and in some cases faster than some cases faster than transcription. It has been reported transcription. It has been reported that in some bacteria an average that in some bacteria an average of 10 amino acid residues are of 10 amino acid residues are added to the peptide chain ever added to the peptide chain ever secondsecond

prof. azaprof. aza

prof. azaprof. aza

11.211.2.. Eukaryotic Cells Eukaryotic Cells

•The initiation of protein synthesis The initiation of protein synthesis in eukaryotic cells follows a in eukaryotic cells follows a different route from that found in different route from that found in prokaryotic cells although it still prokaryotic cells although it still uses a methionine-tRNA to start uses a methionine-tRNA to start the process. the process.

•Eukaryotic mRNAs has no Shine-Eukaryotic mRNAs has no Shine-Dalgarno sequences but are Dalgarno sequences but are characterised by a 7-methyl GTP characterised by a 7-methyl GTP unit at the 5’ end of the mRNA unit at the 5’ end of the mRNA strand and a polyadenosine strand and a polyadenosine nucleotide tail at the 3’ end of nucleotide tail at the 3’ end of the strand (Figure 10.16).the strand (Figure 10.16).

prof. azaprof. aza

prof. azaprof. aza

prof. azaprof. aza

• In eukaryotic cells, the initiating In eukaryotic cells, the initiating tRNA is a unique form of the tRNA is a unique form of the activated methionine- tRNA activated methionine- tRNA (tRNA(tRNAii MetMet). However, unlike in the ). However, unlike in the case of prokaryotic cells, the case of prokaryotic cells, the methionine residue it carries is methionine residue it carries is not formylated. not formylated.

•The initiating process is started by The initiating process is started by this tRNAi Met binding to the 40S this tRNAi Met binding to the 40S subunit of the ribosome to form the subunit of the ribosome to form the so-called preinitiation complex, the so-called preinitiation complex, the process requiring the formation of a process requiring the formation of a complex between tRNAi Met , complex between tRNAi Met , various eukaryotic initiation factors various eukaryotic initiation factors (elFs) and GTP. (elFs) and GTP.

prof. azaprof. aza

•At this point the mRNA binds to the 40S At this point the mRNA binds to the 40S preinitiation complex. This binding process preinitiation complex. This binding process is believed to involve a number of is believed to involve a number of eukaryotic initiation factors and energy eukaryotic initiation factors and energy supplied by the conversions of GTP to GDP supplied by the conversions of GTP to GDP and ATP to ADP. Once the mRNA has and ATP to ADP. Once the mRNA has bound to the preinitiation complex the 60S bound to the preinitiation complex the 60S subunit recombines with the 40S unit to subunit recombines with the 40S unit to form the initiation complex (Figure 10.17).form the initiation complex (Figure 10.17).

prof. azaprof. aza

•Once the mRNA has bound to Once the mRNA has bound to the preinitiation complex the the preinitiation complex the 60S subunit recombines with 60S subunit recombines with the 40S unit to form the the 40S unit to form the initiation complex (Figure initiation complex (Figure 10.17).10.17).

prof. azaprof. aza

•The initiating process is started The initiating process is started by this tRNAi Met binding to the by this tRNAi Met binding to the 40S subunit of the ribosome to 40S subunit of the ribosome to form the so-called preinitiation form the so-called preinitiation complex, the process requiring complex, the process requiring the formation of a complex the formation of a complex between tRNAi Met , various between tRNAi Met , various eukaryotic initiation factors (elFs) eukaryotic initiation factors (elFs) and GTP. and GTP.

prof. azaprof. aza

prof. azaprof. aza

•The absence of the Shine-The absence of the Shine-Dalgarno sequence means that Dalgarno sequence means that an alternative mechanism must an alternative mechanism must he available to align the first he available to align the first AUG codon of the mRNA with the AUG codon of the mRNA with the P site of the ribosome. This P site of the ribosome. This mechanism is believed to direct mechanism is believed to direct the preinitiation complex to the the preinitiation complex to the first AUG codon of the mRNA.first AUG codon of the mRNA.

• Elongation in eukaryotic ribosomes Elongation in eukaryotic ribosomes follows the general mechanism for follows the general mechanism for protein synthesis (see section 10.10.3) protein synthesis (see section 10.10.3) but involves different factors and but involves different factors and proteins from those utilised by proteins from those utilised by prokaryotic ribosomes. Termination only prokaryotic ribosomes. Termination only requires one release factor, unlike in requires one release factor, unlike in prokaryotic ribosomes-where three prokaryotic ribosomes-where three release factors are usually required.release factors are usually required.

prof. azaprof. aza

• Elongation in eukaryotic ribosomes Elongation in eukaryotic ribosomes follows the general mechanism for follows the general mechanism for protein synthesis (see section 10.10.3) protein synthesis (see section 10.10.3) but involves different factors and but involves different factors and proteins from those utilised by proteins from those utilised by prokaryotic ribosomes. prokaryotic ribosomes.

• Termination only requires one release Termination only requires one release factor, unlike in prokaryotic ribosomes-factor, unlike in prokaryotic ribosomes-where three release factors are usually where three release factors are usually required.required.

prof. azaprof. aza

•Termination Termination only requires one only requires one release factor,release factor, unlike in unlike in prokaryotic ribosomes-where prokaryotic ribosomes-where three release factors are three release factors are usually required.usually required.

prof. azaprof. aza

prof. azaprof. aza

Figure 10.17. An outline of the formation of the protein synthesis initiation complex by the ribosomes of eukaryotic cells.