protein synthesis b

8/14/2019 Protein Synthesis b

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Initiation codon

AUG is the most common initiator as it forms the most strongest interaction with

anticodon CAU

GUG and UUG are weak initiators (infCgene coding for IF3 has AUU)

Context of the Start codon

Shine-dalgarno sequence

Kozak sequence


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Shine-dalgarno sequence

3-9 bp long sequence AGGAGGU

Optimally 5 bp upstream of AUG

Activity reduces if about 13 bp upstream to AUG

No activity if farther than 13 bp (even if brought closer by 2o structure)

Base-pairs with 3 sequences of the 16SrRNA. Universally atleast 3 bp pairing is found

Anchors the 30S subunit close to the site of initiation

Prevents reformation of 2o structure in vicinity of the initiation codon

mRNA remains paired with SD even after formation of 1st peptide bond

In absence of SD sequence

If AUG present just at the 5 end of mRNA then low level of translation

Weak initiators (UUG, GUG) cannot initiate even if present at 5 end

However, they may work in coupled translation in multi-cistronicsystems


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Prokaryotes

IF2

Selects and binds fMET-tRNA to 30S subunit

~97 kDa protein coded by infB

GTP binding protein

GTPase activity latent and activated on joining of

50S subunit

Hydrolysis and consequent release of GTP

triggers release of IF2 from the 70S leaving behind

the fMET-tRNA in P site and exposing A site


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Kozak sequence (eukaryotes)

G C C R C C A U GG

Purine at -3, usually A

Most highly conserved

-3 -2 -1 +1 +2 +3 +4

The GCCR region may slow down the scanning thereby facilitating therecognition of AUG


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Leaky scanning

40S by-passes the first AUG and instead initiate at the second and third AUG

Causes:

Lack of good context around 1st AUG

Downstream 20 structures may overcome lack of good context

If 1st AUG too close to the 5 end so cannot be recognized efficiently

If initiation is at a non-AUG codon (CUG, ACG, GUG)

Significance

Means to produce more than one functional protein from 1 mRNA

Regulation of translation


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Reinitiation

80S ribosome translates a small first ORF (upORF)

After termination 40S continues to scan and reinitiates at

downstream AUG

upORF should be small (about 30 codons)

Downstream AUG should be at a distance so that Ifs could be recruited

Probably used to regulate translation


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QuickTime and adecompressor

are needed to see this picture.

Eukaryotic translation initiation factor


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This step involves the eIF4 factors

The 5' end of the mRNA bindseIF4E (cap binding protein) and witheIF4G which may recognise

secondary structure elements downstream of the 5'-end.

PABA (polyA-binding protein), already bound at the 3' end of the mRNA, interacts wit h eIF4G.

The binding ofeIF-4G to PABP also represents a mechanism to ensure that only mature intactmRNAs are translated.

The initiation factors eIF4A and eIF4B join the complex.

eIF4A is an RNA helicase which will remove secondary structure from the mRNA;

eIF4B is an RNA binding protein required for its activ

Preparation of mRNA


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eIF2 (GTP) binds with Met-tRNAiMet

This complex is bound by eIF1-eIF3-eIF5

Finally all are bound to the 40S small subunit of the ribosome


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eIF2 GTP hydrolysis

Hydrolysis of GTP occurs after eIF2 binds to the 30S subunit

Hydrolysis of GTP occurs after scanning subunit reaches AUG

Requires activation by eIF-5 which enters after 30S subunit reaches the AUG

eIF-5 does not induce GTP hydrolysis when contacts in solution but only in presence of

40S

eIF-5 only acts when scanning 30S subunit pauses for long at AUG

Short pauses at others like UUG or CUG are non-productive

GTP hydrolysis triggers a conformational change that releases eIF-2-GDP

eIF-2B required to release GDP

eIF2 phosphorylation

Phosphorylation ofSerine 51 of the alpha subunit of eIF-2

GDP cannot be released from phosphorylated eIF2

Phosphorylated form of eIF2 acts as a competetive inhibitorwith over 150

fold affinity towards eIF2B

Phosph. acts as a mode of regulation for eIF2 activity.


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Elongation


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Aminoacyl-tRNA synthesisAminoacyl-tRNA synthesis

Fidelity of Protein synthesis

Aminoacyl-tRNA synthesisAminoacyl-tRNA synthesis

Codon anticodon recognitionCodon anticodon recognition


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Aminoacyl-tRNA synthetase

Every amino acid has a specific ARS (however there are more than 20 ARSs

Catalyse the esterification of the amino acid and 3 end of tRNA

ARSs are a large family of enzymes

ARSs have active site for recognition of both amino acid and tRNA

The amino acid binding site is well conserved

tRNA binding site is hardly conserved

Two major classes of ARS i.e. Class I and Class II. They differ in active site topologies

ClassI:

Rossmann dinucleotide binding site

Approaches acceptor stem of tRNA from minor grove

ClassII:

Novel antiparallel B sheets

Approaches acceptor stem of tRNA from major grove


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Step1: amino acid and ATP bind the

active sites of the ARS.

Step2: -carboxylate of a.a. attacks -

phosphate of ATP in nucleophile

displacement mecha. To form enzyme

bound aminoacyl-adenylate (anhydride)

with release of PPi.

Step3: 2 or 3 OH at the 3 end of the

tRNA attacks the alhpa-carbonyl of

aminoacyl-adenylate with release of AMP.

Step4: Release of product i.e. aminoacyl-

tRNA


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Recognition of tRNA by AARs


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Aminoacyl end

Anticodon end


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Tu can bind any aminoacylated tRNA except the tRNA-f-Met

Ts is Nucleotide exchange factor

EF-G (eEF2); G-

protein


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Prokaryotes

RF1: Recognizes UAA or UAG

RF2: Recognizes UAA or UGA

Eukaryotes

eRF1: Recognizes all 3 stop codons

RRF: Ribosome release factor


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The word ribosome was coined by Roberts in 1958

Ribosome is a template-directed polymerase, similar in function to an RNA or a DNA polymerase.

The process of Elongation is relatively conserved whereas Initiation and Termination is very variable in

organisms

The A, P and E site are on both the subunits

2.5 x106 in prokaryotes to about 4.5 x106 in higher eukaryotes,two-thirds RNA and one-third protein.

The shape of both the subunits are largely governed by the RNA component

23S rRNA

5S rRNA

35 Proteins

5S rRNA+ Associated

Proteins

Proteins L7 and L12

Proteins L1

Haloarcula marismortui

Base of stalk has the factor-binding site (for all the GTPbinding proteins like EF-Tu, EF-G, IF-2, RF1,2,3)


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Large subunit is Monolithic i.e. no


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23S rRNA11 stem-loops

forming regions

6 domains (stem-loop

with large loop that again

forms stem-loop)

5 stem-loops

5 and 3 ends form a helix which binds the entire molecule.

All known types (10 types) of secondary structures made by RNA are present. (these are conformations conserved in

all RNA molecules such as T-loop, bulged G motif, kink-turn, hook-turn)

Large subunit is Monolithic i.e. no

sub-structural domains

Long Range interactions:

Stabilize tertiary structures

rRNA large enough to form tertiary/quartianary structures. Tetra-loop-tetraloop receptor motif, ribose zipper,

A-minor motif(streach of As and involved in the interaction of tRNAs to A and P sites.


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makes a 7th domain of the large subunit5S rRNA

Proteins

Stabilization of rRNA structure

Interaction with external proteins

Globular domains of the proteins mostly exterior often in gaps and crevices formed by

the rRNAProteins are absent from the active site and the flat surface (where the ribosomal

subunits interact)

All except L12 interacts directly with RNA

L22 interacts with all the 6 domian of rRNA (23S)

Proteins bind to RNA by recognizing the shape of the RNA molecuole as interaction is

via the phosphate backbone

The tails of these protiens are highly basic (1/4th Argenine and Lysine)

The tail sequence is more conserved that the globular regions

Basic nature helps to stabilise RNA

They make the surface of the ribosome ve while interior is +ve


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The tunnel is mainly composed of RNA and only 1-2nm wide and 10nm long. Constrains the

peptide chain so it does not fold before leaving the exit domain

It can hold about 50 a.a.


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The structure of the small subunit (30S) is largely

governed by the 16S rRNA

There is a asymmetrical distribution of RNA and

proteins

The interface where 30S interacts with 50S does not

have any proteins. Thus the interaction is majorly

between 16S and 23S rRNAs

Only regions 2 proteins S7 and S12 lie near the

interface

Protein S1 has strong affinity to single stranded nucleic

acid and required for initial binding of the mRNA. Itkeeps the mRNA as linear molecule. S1 along with S18

and S21 forms the domain that interacts with the mRNA

and initiator tRNA

The 3 end of 16S interacts directly with the mRNA

It also interacts with the anti-codons in both A and P

sites


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eIF4E bnds to the cap and eIF4G and other proteins (eIF4E-binding

protein) thus acts to recruit the complex to the cap

It is the major target for

Conserved tryptophan ring of eIF4E interact directly with the methly groupof the cap


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Elongation

Decoding at P site

Formationof peptide bond

Translocation of peptidal tRNA from the A site


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protein synthesis b

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