Nida Nosher08030614-050

Topic: Amino acids Coding CodonsBs 7th

Presented to: Miss Aqsad

PROTEIN SYNTHESIS

TRANSLATION

The Genetic Code

The nucleotide sequence of mRNA contains three letter codons that specify all of the 20 amino acids found in proteins plus a signal to terminate protein synthesis

The order that the codons appear in the mRNA (5’ - 3’) directly dictates the order of the amino acids in the polypeptide chain of the protein (N - C termini)

Amino Acid DNA Base Triplets M-RNA Codons T-RNA Anticodons

alanine  CGA, CGG, CGT, CGC   GCU, GCC, GCA, GCG   CGA, CGG, CGU, CGC 

arginine GCA, GCG, GCT, GCC

TCT, TCCCGU, CGC, CGA, CGG

AGA, AGGGCA, GCG, GCU, GCC

UCU, UCC

asparagine TTA, TTG AAU, AAC UUA, UUG

aspartate CTA, CTG GAU, GAC CUA, CUG

cysteine ACA, ACG UGU, UGC ACA, ACG

glutamate CTT, CTC GAA, GAG CUU, CUC

glutamine GTT, GTC CAA, CAG GUU, GUC

glycine CCA, CCG, CCT, CCC GGU, GGC, GGA, GGG CCA, CCG, CCU, CCC

histidine GTA, GTG CAU, CAC GUA, GUG

isoleucine TAA, TAG, TAT AUU, AUC, AUA UAA, UAG, UAU

leucine AAT, AAC, GAA, GAG

GAT, GACUUA, UUG, CUU, CUC

CUA, CUGAAU, AAC, GAA, GAG

GAU, GAC

lysine TTT, TTC AAA, AAG UUU, UUC

methionine TAC AUG UAC

phenylalanine AAA, AAG UUU, UUC AAA, AAG

proline GGA, GGG, GGT, GGC CCU, CCC, CCA, CCG GGA, GGG, GGU, GGC

serine AGA, AGG, AGT, AGC

TCA, TCGUCU, UCC, UCA, UCG

AGU, AGCAGA, AGG, AGU, AGC

UCA, UCG

stop ATG, ATT, ACT UAA, UAG, UGA AUG, AUU, ACU

threonine TGA, TGG, TGT, TGC ACU, ACC, ACA, ACG UGA, UGG, UGU, UGC

tryptophan ACC UGG ACC

tyrosine ATA, ATG UAU, UAC AUA, AUG

valine CAA, CAG, CAT, CAC GUU, GUC, GUA, GUG CAA, CAG, CAU, CAC

Genetic code can be read in 3 ways depending upon where you start!

+1 frameshift

+2 frameshift

The genetic information encoded in each reading frame is different

DIF

FE

RE

NT

RE

AD

ING

FR

AM

ES

OF

m

RN

A T

HE

SA

ME

SE

QU

EN

CE

Reading Frames Since codons consist of 3 bases, there

are 3 “reading frames” possible on an RNA (or DNA), depending on whether you start reading from the first base, the second base, or the third base.– The different reading frames give

entirely different proteins.– Consider ATGCCATC, and refer to the

genetic code. (X is junk)• Reading frame 1 divides this into ATG-

CCA-TC, which translates to Met-Pro-X• Reading frame 2 divides this into A-TGC-

CAT-C, which translates to X-Cys-His-X• Reading frame 3 divides this into AT-

GCC-ATC, which translates to X-Ala-Ile • Each gene uses a single reading

frame, so once the ribosome gets started, it just has to count off groups of 3 bases to produce the proper protein.

Transfer RNAs (tRNA) act as adapters between the mRNA and protein synthesising machinery (‘ribosomes’)

How is the mRNA genetic code read during protein synthesis?

tRNA triplet nucleotide sequences that are complementary to mRNA codons, called ‘anticodons’, form specific base-pairs with the mRNA codons

As each specific tRNA (i.e. defined by its anticodon) is bound to a specific amino acid at its 3’ end, according to the genetic code in the mRNA, is recruited to the ribosome

Some tRNA can read more than one codon !

Adenosine to inosine conversion at the wobble position of the anticodon in some

tRNAs permit it to recognise three different codons !

This is because the first base of the anti-codon (that binds to the third base of the mRNA codon) is not squeezed/ constrained as it would be in a DNA double helix and can wobble making other base pairings possible i.e. ‘wobble base-paring‘

The minimum set of required tRNAs is 31 but there are 61 possible amino acid coding codons !

Therefore a single tRNA can two recognise two different codons for the same amino acid !

Figure 6-58 Molecular Biology of the Cell (© Garland Science 2008)

Attachment of amino-acids to tRNAs (‘Charging’)

Each tRNA is charged by a specific enzymes that recognise both the tRNA and the amino acid - called ‘aminoacyl tRNA synthetases‘

e.g. tryptophanyl tRNA synthetase

Charging is a two step process

(Aminoacyl-AMP)

tRNA charging

Uncharged tRNA

Charged tRNA

2. Transfer of the amino acid to the free 3’OH of

the tRNA

1. Amino acid adenylation

amino (N-) terminus carboxyl (C-) terminuspeptide bond

During protein synthesis tRNAs are sequentially released from their corresponding amino acids

Very large protein-RNA complexes called ‘Ribosomes’

Ribosome comprise one large and one small subunit

Ribosomes bind both the mRNA and amino acid charged tRNAs to decode the information in the mRNA into a polypeptide sequence of amino acids

Prokaryotic 16S rRNA

Ribosomal RNA (‘rRNA’) critical to ribosome function

rRNAs:

• 2/3 of the molecular weight for ribosome (prokaryotes)

• form complex and defined secondary structure

• originally thought to have structural role, now known to required for most of the ribosome’s functions

• X-ray crystallography show no proteins are proximal to catalytic site to participate in peptide bond formation

• 23S rRNA (prokaryotes) acts as a ‘peptidyl transferase’ ribozyme

• sequence mutagenesis studies of 23S rRNA show its function is to correctly position the incoming charged tRNA to allow spontaneous formation of the peptide bond

Figure 6-64 Molecular Biology of the Cell (© Garland Science 2008)

3D ribosomal structure (70S prokaryotic)

The interface between large & small s/u’s form a groove for mRNA binding and three tRNA binding sites: A (acceptor), P (peptide) & E (exit)

Prokaryotic ribosomes

nnnnnnAGGAGGUnnnnnnnAUGnnnnnnn UCCUCCA

Shine-Delgarno sequence

start codon

16S rRNA base-pairing leads to small ribosomal s/u recognition, large s/u

recruitment and formation of the ‘70S initiation complex’

Correctly identifying the translation start-point in mRNA

Translation always starts at an AUG codon (coding for methionine) called the ‘start codon’

How does the ribosome

recognise the correct AUG as

the start codon ?

7bp

Shine-Delgarno sequence

Enables translation of polycistronic mRNAs

N-Formyl methionine charged tRNA is then recruited into the P-site ready for translation to start

mRNA

Various ‘initiation

factors (IFs)’ participate in this

process

Variations in the S-D sequence can effect translation initiation efficiency

The elongation phase of translation is essentially similar in prokaryotes

and eukaryotes involving a repetition of a series of steps

Elongation phase of translation

Charged tRNA enters A-site.

Specificity dictated by

codon-anticodon base-pairing

New peptide bond formation

(between adjacent amino acids in P

& A-sites)

Ribosome ‘translocates’ along mRNA to

next codon

Bound tRNAs move to next site (A-P or P-E)

As next charged tRNA enters A-site the E-site occupant

departs the ribosome

The elongation phase is governed ‘elongation factors (EFs)’

Prokaryotic example used below (eukaryotes have other EFs but principle is the same)

‘EF-Tu’ binds to charged tRNAs and delivers them to the A-site. This requires energy from GTP

hydrolysis to GDP

GDP

EF-Ts

‘EF-Ts’ exchanges GDP from EF-Tu for fresh GTP allowing it to recruit more charged tRNAs to the A-

site

Ribosomal TRANSLOCATION along the mRNA and the

associated migration of the t-RNAs from the A- to P-site or P- to E-site

also requires energy from GTP hydrolysis mediated by ‘EF-G’

EF-G binding causes bound tRNAs to exist partially bound to both sites

(A & P or P & E) and GTP hydrolysis completes the

translocation

1. No tRNAs can recognise a stop codon in the A-site. The stop codon is therefore recognised by a ‘release factor (RF)’ (either RF1 or RF2 depending on stop codon

sequence)

Termination of translation

2. RFs activate the peptidyl-transferase of the ribosome to hydrolyse the bond between the completed polypeptide chain and the tRNA in the P-site

3. Further RFs (RF3 and ‘Ribosome recycling factor (RRF)’ dissociate RF1/2 and the small/ large ribosomal s/u’s

Proteins are polymers of different amino acids joined by peptide bonds

Each amino acid has a different chemical side chain and the order of these side chains in a protein sequence is what conveys its structure and functionality

Amino acids

Polypeptide (i.e. protein)

Protein synthesis

Hydrophylic Hydrophobic

Grouping the 20 amino acids by their chemical properties

Learn the amino acid abbreviations and properties

e.g. Heat Shock Protein 70 (Hsp70)

The expression of Hsps (heat shock proteins) increases as temperature increases because folded proteins are more likely to unfold/ denature at higher temperatures

1. Hsp70-ATP able to loosely bind hydrophobic patches of amino acids as they emerge from the

ribosome

2. Peptide binding induces intrinsic ATPase activity in

HSP70

3. Hsp70-ADP tightly associates with unfolded protein and

protects it from aggregating

4. Nucleotide exchange factors eventually replace the ADP with

ATP and HSP70 releases the unfolded protein

5. Protein spontaneously folds into correct confirmation

6. A small percentage of protein incorrectly folds

e.g. GroEL/ Hsp60 ‘rescues’ misfolded proteins

1. Misfolded proteins with exposed hydrophobic regions bind hydrophobic

regions in the neck of the GroEL

Multi-subunit complex ‘cocktail shaker’

2. The binding of the GroES cap andATP cause conformational change that releases the misfolded protein into the lumen where it can fold, sequestered from the

cytoplasm

3. Hydrolysis of the bound ATP (plus binding of additional ATP) releases the GroES cap and the

correctly folded protein

4. Another misfolded protein binds the opposite side of the GroEL complex

Structural proteinsCytoskeletonExtracellualr matrix

Mechanical proteinsactin, myosin

Enzymes

Binding proteinstransport, storage

Information processing proteinsreceptors, signalling

Proteins perform many diverse functions

Proteins are therefore subject to tight regulation to control these functions

Any Question?