Protein Synthesis B

Initiation codon ‘AUG’ is the most common initiator as it forms the most strongest interaction with anticodon ‘CAU’ ‘GUG’ and ‘UUG’ are weak initiators (infC gene coding for IF3 has AUU)

Shine-dalgarno sequence Context of the Start codon Kozak sequence

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-cistronic systems

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

Kozak sequence (eukaryotes)

-3

-2 -1 +1

GCCRCCA

+2 +3

+4

UGG

Purine at -3, usually ‘A’ Most highly conserved The GCCR region may slow down the scanning thereby facilitating the recognition of AUG

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

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

Eukaryotic translation initiation factor

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Preparation of mRNA This step involves the eIF4 factors The 5' end of the mRNA binds eIF4E (cap binding protein) and with eIF4G which may recognise  secondary structure elements downstream of the 5'­end. PABA (polyA­binding protein), already bound at the 3' end of the mRNA, interacts with eIF4G.  The binding of eIF­4G to PABP also represents a mechanism to ensure that only mature intact  mRNAs 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

eIF2 (GTP) binds with Met­tRNAiMet This complex is bound by eIF1­eIF3­eIF5 Finally all are bound to the 40S small subunit of the ribosome

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 of Serine 51 of the alpha subunit of eIF-2 GDP cannot be released from phosphorylated eIF2 Phosphorylated form of eIF2 acts as a competetive inhibitor with over 150 fold affinity towards eIF2B Phosph. acts as a mode of regulation for eIF2 activity.

Elongation

Aminoacyl-tRNA synthesis Fidelity of Protein synthesis Codon anticodon recognition

Aminoacyl-tRNA synthesis

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

Step1: amino acid and ATP bind the active sites of the ARS.

Step2: α-carboxylate of a.a. attacks αphosphate 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. aminoacyltRNA

Recognition of tRNA by AARs

Aminoacyl end

Anticodon end

Tu can bind any aminoacylated tRNA except the tRNA-f-Met Ts is Nucleotide exchange factor

EF-G (eEF2); Gprotein

Prokaryotes RF1: Recognizes UAA or UAG RF2: Recognizes UAA or UGA Eukaryotes eRF1: Recognizes all 3 stop codons RRF: Ribosome release factor

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

Haloarcula marismortui

5S rRNA+ Associated Proteins Proteins L7 and L12

23S rRNA 5S rRNA Proteins L1

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

35 Proteins

Large subunit is ‘Monolithic’ i.e. no sub-structural domains 23S rRNA

11 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) 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 ‘A’s and involved in the interaction of tRNAs to ‘A’ and ‘P’ sites.

5S rRNA

makes a 7th domain of the large subunit

Proteins Stabilization of rRNA structure Interaction with external proteins Globular domains of the proteins mostly exterior often in gaps and crevices formed by the rRNA Proteins 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

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.

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. It keeps 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

Active sites of the ribosome are not small discrete regions but large structures The tRNA in the ‘A’ and ‘P’ sites are nearly parallel to each other The anticodon loop of the tRNAs bind to the domain in the small subunit while rest of the body is bound to the 50S

26o

The 3’ ends of the tRNAs are the closest (5Å) to facilitate transfer of the peptidal chain. The mechanism of catalysis is largely entropic i.e. positioning of the substrate, desolvation and electrostatic shielding (in contrast to enthalpic such as acid-base catalysis or covalent catalysis used by most other enzymes). 45o kink

Concerted proton-shuttle mechanism. The P-site and A-site tRNA substrates are blue and red, espectively, ribosome residues are green, and ordered water molecules that stabilize the developing charges are gray. The attack of the a-NH2 group on the ester carbon results in a six-membered transition state, in which the 20-OH group of the A-site A76 ribose moiety donates its proton to the adjacent 30 oxygen while simultaneously receiving one of the amino protons [9,42]. Alternatively, the water molecule (*) might be used for a proton shuttle.

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 group of the cap

Elongation Decoding at ‘P’ site Formationof peptide bond Translocation of peptidal tRNA from the ‘A’ site