Molecular biology

Field of science concerned with the

chemical structures and processes of

biological phenomena at the molecular

level. Having developed out of the

related fields of biochemistry,

genetics, and biophysics, the

discipline is particularly concerned

with the study of proteins, nucleic

acids, and enzymes. In the early

1950s, growing knowledge of the

structure of proteins enabled the

structure of DNA to be described.

The discovery in the 1970s of certain

types of enzymes that can cut and

recombine segments of DNA

(recombination) in the chromosomes of

certain bacteria made recombinant-DNA

technology possible. Molecular

biologists use that technology to isolate

and modify specific gene.

• Nucleic acids and nucleoprotein structure.

• Replication.

• Transcription.

• Regulation of gene expression.

• Restriction enzymes & its function in DNA technology.

• Gene cloning .

• Production of recombinant plasmid.

• Construction of genomic and DNA libraries.

• Analyzing & sequencing cloned DNA.

• Analysis of specific nucleic acids in complex mixtures

• polymerase chain reaction (PCR),mutation .

Deoxyribonucleic acid

• In living organisms, DNA does not usually

exist as a single molecule, but instead as

a tightly-associated pair of molecules.

These two long strands entwine like vines,

in the shape of a double helix. The

nucleotide repeats contain both the

segment of the backbone of the molecule,

which holds the chain together, and a

base, which interacts with the other DNA

strand in the helix.

In general, a base linked to a sugar is called a

nucleoside and a base linked to a sugar and one

or more phosphate groups is called a

nucleotide. If multiple nucleotides are linked

together, as in DNA, this polymer is referred to

as a polynucleotide. Nucleic acids are polymeric

macromolecules made from nucleotide monomers. In

DNA, the purine bases are adenine and guanine, while

the pyrimidines are thymine and cytosine. RNA uses

uracil in place of thymine.

• Nucleotide structure

• A nucleotide is composed of a nucleobase

(nitrogenous base), a five-carbon sugar (either

ribose or 2'-deoxyribose), and one to three

phosphate groups. Together, the nucleobase and

sugar comprise a nucleoside. The phosphate

groups form bonds with either the 2, 3, or 5-carbon

of the sugar, with the 5-carbon site most common.

Cyclic nucleotides form when the phosphate group

is bound to two of the sugar's hydroxyl groups.

Ribonucleotides are nucleotides where the sugar is

ribose, and deoxyribonucleotides contain the sugar

deoxyribose. Nucleotides can contain either a

purine or pyrimidine base.

Synthesis

Nucleotides can be synthesized by a variety of means

both in vitro and in vivo. In vivo, nucleotides can be

synthesised de novo or recycled through salvage

pathways. Nucleotides undergo breakdown such that

useful parts can be reused in synthesis reactions to

create new nucleotides. In vitro, protecting groups may

be used during laboratory production of nucleotides. A

purified nucleoside is protected to create a

phosphoramidite, which can then be used to obtain

analogues not found in nature and/or to synthesize an

oligonucleotide

DNA's duplex nature • DNA is normally double-stranded. The sequences of

the two strands are related so that an A on one strand is matched by a T on the other strand; likewise, a G on one strand is matched by a C on the other strand. Thus, the fraction of bases in an organism's DNA that are A is equal to the fraction of bases that are T, and the fraction of bases that are G is equal to the fraction of bases that are C. For example, if one-third of the bases are A, one-third must be T, and because the amount of G equals the amount of C, one-sixth of the bases will be G and one-sixth will be C. The importance of this relationship, termed Chargraff's rules, was recognized by Watson and Crick, who proposed that the two strands form a double helix with the two strands arranged in an antiparallel fashion, interwound head-to-tail

• In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand. This arrangement of DNA strands is called antiparallel. The asymmetric ends of DNA strands are referred to as the 5′ (five prime) and 3′ (three prime) ends.

• One of the major differences between DNA and RNA is the sugar, with 2-deoxyribose being replaced by the alternative pentose sugar ribose in RNA.

• Usually,we read nucleic acid sequences of DNA in a 5′ to 3′ direction, so a DNA dinucleotide of (51) adenosine-guanosine (31) is read as AG.

• The complementary sequence is CT, because both sequences are read in the 5′ to 3′ direction. The terms 5′ and 3′ refer to the numbers of the carbons on the sugar portion of the nucleotide (the base is attached to the 1′ carbon of the sugar).

• Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information.

Nucleotides

Adenosine monophosphate

AMP

Adenosine diphosphate

ADP

Adenosine triphosphate

ATP

Guanosine monophosphate

GMP

Guanosine diphosphate

GDP

Guanosine triphosphate

GTP

Thymidine monophosphate

TMP

Thymidine diphosphate

TDP

Thymidine triphosphate

TTP

Deoxynucleotides

Deoxyadenosine monophosphate

dAMP

Deoxyadenosine diphosphate

dADP

Deoxyadenosine triphosphate

dATP

Deoxyguanosine monophosphate

dGMP

Deoxyguanosine diphosphate

dGDP

Deoxyguanosine triphosphate

dGTP

thymidine monophosphate

TMP

thymidine diphosphate

TDP

thymidine triphosphate

TTP

Deoxyuridine monophosphate

dUMP

Deoxyuridine diphosphate

dUDP

Deoxyuridine triphosphate

dUTP

Deoxycytidine monophosphate

dCMP

Deoxycytidine diphosphate

dCDP

Deoxycytidine triphosphate

dCTP

Pyrimidine ribonucleotides

Pyrimidine nucleotide synthesis starts with the formation of carbamoyl phosphate from glutamine and CO2. The cyclisation reaction between carbamoyl phosphate reacts with aspartate yielding orotate in subsequent steps. Orotate reacts with 5-phosphoribosyl α-diphosphate (PRPP) yielding orotidine monophosphate (OMP) which is decarboxylated to form uridine monophosphate (UMP). It is from UMP that other pyrimidine nucleotides are derived. UMP is phosphorylated to uridine triphosphate (UTP) via two sequential reactions with ATP. Cytidine monophosphate (CMP) is derived from conversion of UTP to cytidine triphosphate (CTP) with subsequent loss of two phosphates

Nucleotides function in cell metabolism Purine ribonucleotides

The atoms which are used to build the purine nucleotides come from a variety of sources:

The de novo synthesis of purine nucleotides by which these precursors are incorporated into the purine ring, proceeds by a 10 step pathway to the branch point intermediate IMP, the nucleotide of the base hypoxanthine. AMP and GMP are subsequently synthesized from this intermediate via separate, two step each, pathways. Thus purine moieties are initially formed as part of the ribonucleotides rather than as free bases.

Synthesis Purine ribonucleotides By using a variety of isotopically labeled

compounds it was demonstrated that the sources of the atoms in purines are as follows:

The biosynthetic origins of purine ring atoms N1 arises from the amine group of Asp C2 and C8 originate from formate N3 and N9 are contributed by the amide group of Gln C4, C5 and N7 are derived from Gly C6 comes from HCO3

- (CO2)

DNA is a long polymer made from repeating units called nucleotides.[The DNA chain is 22 to

26 Angstroms' wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Ångstroms

(0.33 nanometres) long. Although each individual repeating unit is very small, DNA polymers can be

enormous molecules containing millions of nucleotides. For instance, the largest human

chromosome, chromosome number 1, is 220 million base pairs long.

Major and minor grooves

The double helix is a right-handed spiral. As the DNA strands wind around each other, they leave gaps between each set of phosphate backbones, revealing the sides of the bases inside

Two of these grooves twisting around the surface of the double helix: one groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove

Base pairing

Each type of base on one strand forms a bond with just one type of base on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with A bonding only to T, and C bonding only to G. This arrangement of two nucleotides binding together across the double helix is called a base pair. In a double helix, the two strands are also held together via forces generated by the hydrophobic effect and pi stacking, which are not influenced by the sequence of the DNA. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in living organisms.

The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds. The GC base pair is therefore stronger than the AT base pair. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determine the strength of the association between the two strands of DNA.

Long DNA helices with a high GC content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands.

Parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in bacterial promoters, tend to have sequences with a high AT content, making the strands easier to pull apart.

Sense and antisense

A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is complementary to the sense sequence and is therefore called the "antisense" sequence. Since RNA polymerases work by making a complementary copy of their templates, it is this antisense strand that is the template for producing the sense messenger RNA. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences).

Biological molecules that prefer to form strands. Wilkins worked on the DNA project with Rosalind Franklin, who took the X-ray photograph that gave Watson and Crick their eureka moment. He then spent almost 10 years rigorously verifying that breakthrough.

Linking number : in topology, the total number of times one strand of the DNA double helix winds around the other in a right hand direction, given a DNA molecule with constrained ends. 2 molecules differing only in linking number are topoisomers.

Writhing number (W) : in topology, the number of superhelical turns in a DNA molecule with constrained ends

Alternative double-helical structures DNA exists in several possible conformations.

The conformations so far identified are: A-DNA, B-DNA, C-DNA, D-DNA, E-DNA,H-DNA, L-DNA, P-DNA, and Z-DNA

However, only A-DNA, B-DNA, and Z-DNA have been observed in naturally occurring biological systems

Which conformation DNA adopts depends on the sequence of the DNA, the amount and direction of supercoiling, chemical modifications of the bases and also solution conditions, such as the concentration of metal ions and polyamines

•The A -DNA is a wider right-handed spiral, with a shallow and wide minor groove and a narrower and deeper major groove. The A form occurs under non-physiological conditions in dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, as well as in enzyme-DNA complexes

•B-DNA : the usual double helical structure assumed by double-stranded DNA; see illustration at deoxyribonucleic acid. •Z-DNA : a form of DNA in which the phosphate groups form a dinucleotide repeating unit zigzagging up a left-handed helix with a single, deep groove; it is particularly likely to occur in stretches of alternating purines and pyrimidines From left to right, the structures of A, B

and Z DNA

•spacer DNA : the nucleotide sequences occurring between genes, in eukaryotes often long and including many repetitive sequences; particularly, the DNA occurring between the genes encoding ribosomal RNA.

•complementary or copy DNA (cDNA) : synthetic DNA transcribed from a specific RNA through the reaction of the enzyme (reverse transcriptase). •nuclear DNA (nDNA) : the DNA of the chromosomes found in the nucleus of a eukaryotic cell.

Repetitive DNA : nucleotide sequences occurring multiply within a genome; they are characteristic of eukaryotes and generally do not encode polypeptides. Sequences may be clustered or dispersed, and repeated moderately (10 to 104 copies per genome) to highly (>106 copies per genome). Moderately repetitive DNA sequences encode some structural genes for ribosomal RNA and histones; highly repetitive sequences are mostly satellite DNA

Satellite DNA : short, highly repeated DNA sequences found in eukaryotes, usually in clusters in constitutive heterochromatin and generally not transcribed

Mitochondrial DNA (mtDNA) : the DNA of the mitochondrial chromosome, existing in several thousand copies per cell and inherited exclusively from the mother. Its code differs both from that of nuclear DNA and from that of any present day prokaryote, and it evolves 5 to 10 times more rapidly than nuclear DNA.

Recombinant DNA : a DNA molecule composed of

linked sequences not normally occurring within the same molecule, such as a bacterial plasmid into which has been inserted a segment of viral DNA.

Single copy DNA (scDNA) : nucleotide sequences

present once in the haploid genome, as are the majority of the gene sequences encoding polypeptides in eukaryotes

THE CENTRAL DOGMA OF MOLECULAR BIOLOGY

( THE BIOINFORMATION

THEORY)

Central Dogma of Biology

Replication • Replication • Chromosomes are located in the nucleus of a cell. DNA

must be duplicated in a process called replication before a cell divides. The replication of DNA allows each daughter cell to contain a full complement of chromosomes.

• DNA Replication: • Semiconservative Model of DNA Replication After Watson and Crick proposed the double helix

model of DNA, three models for DNA replication were proposed: conservative, semiconservative, and dispersive. The semiconservative model was proved to be the correct one

Semiconservative DNA replication The two strands in the double helix separate, and then each strand serves as template for the synthesis of a new (complementary) strand. After replication has been completed, each of the two duplexes has one old and one newly synthesized strand. and dispersive modes of replication do not make much sense, and are not supported by experiments.

Eukaryotic DNA replication is semiconservative

Eukaryotic DNA replicates Semiconservatively

by the Taylor, Woods, and Hughes experiment in 1958.

They labeled DNA with 3H-T, treated the roots of Fava bean with Colchicin, fixed and prepared for microscopy. At the first metaphase, after labeling at interphase, both chromatids of each chromosome ere labeled, whereas at the second metaphase only one chromatide was labeled

How does this show semiconservative replication?

The DNA double helix and genetic

replication • Because an A on one strand must base-pair with a T on

the other strand, if the two strands are separated, each single strand can specify the composition of its partner by acting as a template.

• The DNA template strand does not carry out any enzymatic reaction but simply allows the replication machinery (an enzyme) to synthesize the complementary strand correctly.

• This dual-template mechanism is termed semi-conservative, because each DNA after replication is composed of one parental and one newly synthesized strand. Because the two strands of the DNA double helix are interwound, they also must be separated by the replication machinery to allow synthesis of the new strand. Figure 3 shows this replication.

Features of DNA replication • Bidirectional. Starts at specific sites (origins) and moves • in opposite directions using two replication “forks”. • • Semi-discontinuous. One strand (leading) replicates continuously and the other (lagging) discontinuously • • In the 5’ - 3’ direction. Enzymes (DNA polymerases) can

only add a nucleotide to a free OH group at the 3'-end of a growing chain

The double-stranded DNA shown above is unwinding and ready for

replication. Note the antiparallel nature of the strands; that is, the 5'

to 3' orientation of the top strand and the 3' to 5' orientation of the

complementary bottom strand.

A. The DNA is already partially unwound to form a replication fork. B. On the bottom template strand, primase synthesizes a short RNA primer in the 5' to 3' direction. C. Primase leaves, and DNA polymerase adds DNA nucleotides to the RNA primer in the 5' to 3' direction. In E. coli the enzyme used is DNA polymerase III. This new DNA is called the leading strand because it is being made in the same direction as the movement of the replication fork.

Enzymes and Proteins in DNA Replication

• A large number of enzymes and other proteins are involved in the synthesis of new DNA at a replication fork.

• Alternative DNA polymerase:

• This DNA polymerase replaces the RNA primer with DNA. This is a different type of DNA polymerase from the main DNA polymerase which synthesizes DNA on a DNA template.

• In E. coli the main enzyme is DNA polymerase III and the enzyme that replaces the RNA primer with DNA is DNA polymerase I.

• When the RNA primer has been replaced with DNA, there is a gap between the two Okazaki fragments and this is sealed by DNA ligase

DNA ligase: • DNA ligase seals the gap left between Okazaki fragments

after the primer is removed. As the Okazaki fragments are joined, the new lagging strand becomes longer and longer.

• DNA polymerase: • Location: On the template strands. • Function: Synthesizes new DNA in the 5' to 3' direction using

the base information on the template strand to specify the nucleotide to insert on the new chain. Also does some proofreading; that is, it checks that the new nucleotide being added to the chain carries the correct base as specified by the template DNA. If an incorrect base pair is formed, DNA polymerase can delete the new nucleotide and try again.

• • Lagging Strand: • The new DNA strand made discontinuously

in the direction opposite to the direction in which the replication fork is moving.

• • Leading strand: • The new DNA strand made continuously in

the same direction as movement of the replication fork.

• • Okazaki fragment: • Location: On the template strand which

dictates new DNA synthesis away from the direction of replication fork movement.

• Function: A building block for DNA synthesis of the lagging strand. On one template strand, DNA polymerase synthesizes new DNA in a direction away from the replication fork movement. Because of this, the new DNA synthesized on that template is made in a discontinuous fashion; each segment is called an Okazaki fragment.

• • Helicase: • Location: At the replication fork.

• Function: Unwinds the DNA double helix.

• • Primase: • Location: Wherever the synthesis of a

new DNA fragment is to commence. Function: DNA polymerase cannot start the synthesis of a new DNA chain, it can only extend a nucleotide chain primer. Primase synthesizes a short RNA chain that is used as the primer for DNA synthesis by DNA polymerase.

• Single-strand binding (SSB) proteins:

Location: On single-stranded DNA near the replication fork. Function: Binds to single-stranded DNA to make it stable.

• Overall direction of

replication (movement

of replication fork):

The direction of replication i.e.,

the direction in which the

replication fork moves as the

DNA double helix unwinds.

• Parent DNA:

The parental DNA double helix

that will be unwound and used

as the template for new DNA

synthesis.