gene expression…from DNA to protein

biology 1

• Genes control metabolism• Gene expression is a two stage process

– transcription– translation

• Genes consists of triplets of nucleotides - the genetic code

• Protein synthesis in prokaryotes and eukaryotes– Eukaryotic modification of RNA

• Mutations

Genes control metabolism• One gene-One polypeptide rule• Polypeptides that are constructed as a result of

transcription/translation process become either– structural proteins– enzymes

• Those proteins that have quaternary structure may have polypeptides originating from different genes

The transcription/translation process

• Transcription: DNA codes for the construction of mRNA

• Translation: mRNA is read by rRNA at a ribosome; tRNA brings amino acids to ribosome as defined by code on mRNA

• Ribosome assembles polypeptide

Recap on RNA - a ribose nucleic acid that uses Uracil (U) in place of Thymine (T)

The genetic code• The linear sequence of nucleotides in DNA ultimately

determines the linear sequence of amino acids in a polypeptide• There are approximately 20 types of amino acid to choose from• In DNA, the four nucleotides are ATCG• Therefore, the sequence of four possible nucleotides must

code for 20 amino acids– If DNA used a individual nucleotide to refer to an individual amino acid,

this system would only code for 41 amino acids– Using two nucleotides would account for 42 = 16 amino acids– Using three nucleotides would account for 43 = 64 amino acids

• Since there are only 20 amino acids, yet 64 possible codes, some redundancy occurs

• Each block of three nucleotides, ultimately corresponding to a particular amino acid, is called a codon

• In the first stage of the gene expression process, transcription, the information in the codons of a gene are transferred to mRNA

• This process is via an RNA polymerase that uses one of the DNA strands of the double helix (the template strand)

• For each amino acid, there are generally several codons possible. Also, some codons have a non-amino acid equivalent, but instead send specific messages to RNA polymerase (start/stop)

Transcription

• Three phases– Polymerase binding and initiation– Elongation– Termination

• In eukaryotes, RNA polymerase II bind to specific regions on DNA called promoters

• Promoters are typically 100 nucleotides long, including– The initiation site, where transcription begins– Nucleotides sequences that help initiate transcription

• Initiation in eukaryotes requires transcription factors, DNA-binding proteins that bind to specific nucleotide sequences in the promoter region– A common place for a transcription factor

to bind is the TATA box– RNA polymerase recognizes the promoter

site once DNA and transcription factor have bound at the TATA box

• RNA polymerase temporarily separates the double helix for transcription

• In elongation, RNA polymerase II (eukaryotes)– Untwists the DNA molecule– Adds incoming RNA free-floating nucleotides to the

3’ end of the RNA strand (grows 5’ to 3’)• mRNA grows at 30-60 nucleotides/sec. The

mRNA chain starts to peel away as the double helix reforms– Followed in series, several molecules of RNA

polymerase can simultaneously transcribe the same gene

• Transcription proceeds until the polymerase reaches a termination code

Translation• During translation, proteins are synthesized according to a

genetic message of sequential codons along mRNA• tRNA (transfer RNA) interprets between the base

sequence in mRNA and the amino acid sequence in a polypeptide chain. To do this…– Transfer amino acids from cytoplasm to ribosome– Recognize the correct codons on mRNA

• Molecules of tRNA are specific to one particular amino acid– One end of tRNA attaches to a specific amino acid (3’ end)– The other end attaches to an mRNA codon by base pairing with

its anti-codon

• An anti-codon is a nucleotide triplet in tRNA• tRNA decodes the genetic message codon by

codon• There are 45 types of tRNA, which is sufficient

for the 64 codes, since there is a relaxation of base-pairing on the third nucleotide (wobble)– e.g., U in 3rd position of anticodon can bind with A

or G on the equivalent codon– In some cases, third position on a tRNA anticodon

is occupied by Inosine (a sixth nucleotide) that can bind with U, C or A

• Joining of tRNA to specific amino acid at the 3’ end is by Aminoacyl-tRNA synthetase

• Each amino acid has a particular synthetase enzyme– ATP activates the amino acid by losing 2 phosphate

groups, and joining to the amino acid as AMP– tRNA bonds to the amino acid, which loses AMP

• Ribosomes coordinate the pairing of tRNA anticodons to mRNA codons– Consist of 2 subunits (small and large) that remain

separated when not involved in protein synthesis– Ribosomes are composed of 60% rRNA and 40%

protein

• In addition to an mRNA binding site, two further sites on a ribosome are the P- and A-sites– P-site holds the tRNA carrying the growing

polypeptide chain– A-site holds the tRNA that has the next amino

acid in the polypeptide sequence• Building of a polypeptide chain consists of

three steps– Initiation– Elongation– Termination

Translation Initiation• In eukaryotes, the small ribosomal unit binds to

an initiator tRNA (methionine; anticodon UAC)• The small ribosomal unit binds to the 5’ end of

mRNA, and in doing so brings the tRNA anticodon in close proximity with mRNA methionine codon

• This binding requires initiation factors• Finally, the large subunit binds to the complex

– The initiator tRNA fits to the p-site of the ribosome– The vacant a-site is ready for the next aminoacyl-tRNA

complex

Translation elongation• Codon recognition—mRNA codon in the a-site of the

ribosome forms hydrogen bonds with anti-codon of an entering tRNA carrying the next amino acid in the chain

• Peptide bond formation—The enzyme peptidyl transferase (part of the large ribosomal unit) catalyzes the peptide bond between the incoming amino acid and the growing polypeptide chain

• Translocation—the tRNA in the p-site releases from the ribosome, and the tRNA in the a-site moves into the vacated site

Translation termination• A termination codon signals the end of translation;

by binding to a protein release factor, this causes:– Peptidyl transferase hydrolyzes the bond between the

completed polypeptide and the tRNA in the p-site– This frees the polypeptide and tRNA so that they can

release from the ribosome– The two ribosomal units disassociate– mRNA may continue to be translated by

polyribosomes

Differences between prokaryotic and eukaryotic gene expression

• Lack of nuclear membrane in prokaryotes means that transcription can occur at one end of the mRNA molecule, while translation can be occurring at the other end

• In eukaryotes, RNA is modified following transcription before translation– 5’ cap added (modified guanine nucleotide– Poly-A tail added (200 adenine nucleotides) to 3’ end– These ends might protect mRNA sequence (attaching to

untranslated leader and trailer sequences respectively)• Gene splicing

Gene splicing• Eukaryotic mRNA has segments of non-code, called introns

(code sequences called exons)– Introns and exons are initially coded into one long strand called hnRNA

(heterogenous RNA)– In RNA splicing, introns are removed from hnRNA to make mRNA

• Process of splicing mRNA involves SnRNPs (“snurps”) - small nuclear ribonucleoproteins, that are composed of SnRNA (small nuclear RNA) and proteins– Together with extra proteins, SnRNPs form complexes called

spliceosomes, which excise introns (SnRNPs attach to either end of each intron)

– tRNA and rRNA also need to be spliced, but different agents do the splicing - ribozymes, RNA molecules that act as enzymes (note: thus not all enzymes are proteins)

• Why do introns exist?– May regulate gene activity– Splicing may regulate export of mRNA to

cytoplasm– Introns cause exons to be further apart,

and therefore to be further away from each other on the chromosome: this could mean a higher probability of recombination during cross-over

– Specific introns may code for specific domains within a protein

When things go wrong...

• Mutation = a permanent change in DNA that can involve large chromosomal regions or a single nucleotide pair

• Point mutation = a mutation limited to one or two nucleotides in a single gene– Base-pair substitution

• Missense mutation• Nonsense mutation

– Insertion/deletion mutations

• Base-pair substitutions generally have no effect if they occur on the third nucleotide of a triplet– If they do change the amino acid, one a.a.

substitution may not radically affect the functionality of the final polypeptide

– In some cases, functionality is improved: in most cases, functionality is impaired

– In nonsense mutations, the substitutions causes a triplet to read STOP, abruptly terminating polypeptide chain. Such mutations are usually harmful

• Insertions or deletions add or remove one or more nucleotides from a sequence– Since a reading frame for nucleotides is

based on a series of three, insertions and deletions that add or remove a sequence of nucleotides not divisible by 3 can substantially alter the final polypeptide

– Such a mutation is referred to as a frameshift - these mutations usually result in non-functional proteins, unless they occur towards the end of a sequence