lecture 7-3

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7. Expression of Genetic Information (3)

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The Genetic Code20 different 4 different nucleotides

Requires at least a range of combinations of 3 nucleotides – 43 – gives 64 possible combinations

If 64 combinations specify 20 What is the function of the remaining 44 codes

Some are specified by more than one codon

A degenerative code

Encoding Genetic Information

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The Genetic CodeThe code is highly degenerate

Nearly all codes specify s

Those that do not are stop codons (3 of the 64)Cause reading of the message to stop

For all organisms – the same codons specify the same s

Exception – codons of mitochondrial mRNAs


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The Genetic Code


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The Genetic CodeChart shows assignments

Non-randomTend to be clustered

Reflects similar codons specifying the same

Spontaneous mutations causing a single base changeMay not cause an change

Similar are specified by similar codonsGreatest similarities in first two nucleotides

eg glycine – 4 codons – all GGX

Greatest variability in third nucleotide of the triplet


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The Genetic Code


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Decoding – Transfer RNAstRNAs act like adaptors

Each tRNA linked to a specific Able to recognize a particular codon of mRNA


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Decoding – Transfer RNAs – StructureAll 73 to 93 nucleotidesUnusual bases

Enzymatic modification of bases after incorporation into the tRNA chain – posttranscriptionallyStructure disrupts H-bonding

Recognition sites for proteins in loop structures

Strings of complementary sequencesFolded into double strand structureIn 2 dimensions appears as a ‘clover leaf’

is attached to the 3’ adenine


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Decoding – Transfer RNAs – Structure

tRNAs fold into a defined tertiary structure

L shapeEach has unique features


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Decoding – Transfer RNAs – StructuretRNA – mRNA complementary base pairing facilitates translation

Interacting tRNA domainThree nucleotides termed the anticodon

Located in the middle loop

Loop contains seven nucleotides – anticodon middle three

Opposite end of molecule from attachment


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Decoding – Transfer RNAsmRNA codons

first two nucleotides – greatest similarities

Third nucleotide – greatest variation

Crick proposed the wobble hypothesisSame tRNA recognizes more than one codon

Rules of wobble at third positionU of anticodon – pairs with A or G of mRNA

G of anticodon – pairs with U or C of mRNA

I (inosine) – pairs with U, C or A of mRNA


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Decoding – Transfer RNAs – activations are covalently linked at the 3’ end of tRNA

Enzyme – aminoacyl-tRNA synthase

Each recognized by a specific aminoacyl-tRNA synthase

Aminoacyl-tRNA synthases – two step reaction:

ATP + aminoacyl-AMP + PPi

Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP


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Decoding – Transfer RNAs – activationAminoacyl-tRNA synthases – two step reaction:

ATP + aminoacyl-AMP + PPi

Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP


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Translating genetic informationMost complex synthetic activity in the cellTranslation in bacterial cellsSimilar in Eukaryotic cells

Difference – translation in eukaryotic cells – a larger number of soluble (non-ribosomal) protein factors

Synthesis – three distinct activitiesInitiation ElongationTermination


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Translating genetic informationInitiation

Ribosome moves along mRNA from one codon to next

To ensure proper triplets are readRibosome attaches at a precise site – the initiation codon

AUG

Ribosome locked into proper reading frame

Mechanism described in a series of steps ---


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Step 1 – Small ribosomal subunit – initiation codon interaction

Binding of small ribosomal subunit to first AUG

Bacterial mRNAs a specific sequence of nucleotides

Shine-Delgarno sequence

5 to 10 nucleotides before initiation sequence

Complementary to a sequence of nucleotides near the 3’ end of bacterial small subunit


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Translating genetic information

InitiationStep 1 – Small ribosomal subunit – initiation codon interaction

Attachment via this interaction in complementary sequences

Initiation factors also involved


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Step 2 – first -tRNA brought to ribosomeAUG also codes for methionine

Always the first Two methionyl-tRNAs

Initiator of protein synthesis – tRNAiMet

General methionyl t-RNA – tRNAMet

tRNAiMet enters complex by binding to AUG and initiation factor

(IF2)


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Step 3 – Assembling initiation complexLarge ribosomal subunit joins the complex

GTP bound to IF2 is hydrolyzed

Release of IF2-GDP


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Translating genetic informationRole of the ribosome

A molecular motor (kinesin and dynein)

During translationRepetitive cycle of mechanical changes

Driven by energy release of GTP hydrolysis

Ribosomal RNAs play a major role in selecting tRNAsAccurate translation

Polymerization of


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Ribosome has three sites for association with tRNAsA site – aminoacyl site

P site – peptidyl site

E site – exit site

tRNAs bind these sites – gap between ribosomal subunits


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Role of the ribosomeInterface contains binding sites for mRNA and incoming tRNA

Catalytic portion of large subunit in a deep cleft – hydrophobic

Tunnel through large subunit – translocation of peptide

RNA associated proteins stabilize the tertiary structure


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Translating genetic informationElongation

Step 1 – Aminoacyl-tRNA selectiontRNAi

Met is in place at the P site

‘A’ site is available for entry of the next -tRNA

Before binding of the -tRNA to the ribosome - it must first bind to a protein elongation factor - EF-Tu

EF-Tu is GTP-linked

EF-Tu delivers the -tRNA to the ribosomal A binding site


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Step 2 – Peptide bond formationAt end of step 1- -tRNA #1 and #2 juxtaposed for reaction

Amino group of at the A site reacts with the carboxyl group of the at the P site

Peptide bond formation occurs spontaneously (no energy input)

Catalysed by peptidyl transferase

Component of the large ribosomal subunit

Peptidyl transferase is a ribozyme


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Step 3 – TranslocationFollowing formation of first peptide bond – tRNA at the A site is bound to a dipeptide – and to mRNA

The tRNA on the P site is devoid of an In translocation – the ribosome and mRNA move relatively

Ribosome moves 3 nucleotides (one codon) along mRNA in the 5’ to 3’ direction

Accompanied by movement of the tRNA dipeptide from the A to the P site

The deacylated tRNA moves from the P site to the E site

Translocation promoted by a GTP-bound elongation factor (EF-G in prokaryotes, eEF2 in eukaryotes)


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Step 4 – Release of deacylated tRNADeacylated tRNA leaves the ribosome – emptying the E site

Each cycle of elongation uses 2 GTP 1 in aminoacyl tRNA selection

1 in translocation

Once peptidyl-tRNA has moved to the P site – the A site is again vacant and ready for entry of another aminoacyl-tRNA


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Step 1

Step 2

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Step 3

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Translating genetic informationTermination

No tRNAs exist whose anticodons are complementary to a stop codon

mRNA stop codons UAA, UAG and UGASignal is read to stop further elongation and release the polypeptide associated with the last tRNA

Termination requires the presence of release factorsBacteria have 3 – RF1, RF2 and RF3

Eukaryotes have 2 – eRF1 and eRF3

Work together to recognize all stop codons

An example of molecular mimicry

Release factor proteins resemble a tRNA


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Release factors enter the A site

A tripeptide in the release factor substitutes for the anticodon of tRNA and interacts directly with the stop codon

Release factor 3 carries a bound GTP which is hydrolysed

Once translation stopsPeptide severed from attachment to last tRNA in the P site

Both release factor and deacylated tRNA are released from the ribosome

Ribosome then separates from mRNA and dissociates into small and large subunits


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Translating genetic information - Overview


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Translating genetic informationPolyribosomes

During translation multiple ribosomes are attached along the mRNA thread

Complex is a polyribosome or polysome

Each ribosome assembles at the initiation codon

Moves toward the 3’ end of mRNA

Simultaneous translation greatly increases the rate of protein synthesis


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lecture 7-3

Documents

trna domain

different nucleotides

nucleotides anticodon

particular codon of

pp i aminoacylamp

c of mrna

codon adegenerativecode

g of mrna g of anticodon