5.9. IRES internal ribosome entry site
RNA structure that allow for translation initiation in the middle of a mRNA sequence Internal ribosomal entry site (IRES), it is a alternative method of initiation of protein synthesis which used particularly by certain viral RNAs, in which a 40S subunit attached straight with IRES.In this situation AUG codons that present at the 5' untranslated region are bypassed completely. In this process the initiation start directly with 40S-mRNA binding which having a complex holding initiator factors and charge initiator tRNA. It was first discovered in picornavirus infection. This virus target eIF4G which binds to cap protein eIF4E. Thus inhibiting the binding of initiation factor with cap by demolish cap structure and prevent host translation process but allow the translation of viral mRNA through IRES.
There are three IRES on the base of how they mediate interaction with 40 S subunit, all three of them posses sequence homology.
- One type of IRES possess initiation codon (AUG) at its upstream periphery. Thus 40S subunit directly interact to it and utilize same factors that are involve for initiation at 5 ' ends.
- Another type presents 100 nucleotide upstream from AUG and 40 S reach up to it by scanning process.
- Hepatitis contain unique type of IRES, here 40 S directly binds to it without requirement of any initiation factor.
5.10. Translational proofreading :- To improve the accuracy of translation's proofreading occurs. It is of tow types.
5.10.1. Chemical proofreading – A proofreading mechanism in which the correction event occurs after the addition of an incorrect subunit to a polypeptide chain, by means of reversing the addition reaction or hydrolyze incorrectly formed aminoacyl adenylates and aminoacyl-tRNAs. Like in case of tyrosine and phenylalanine, both looks similar but tyrosine aaRS form strong hydrogen bonding with hydroxyl group discriminate them. aaRS has catalytic pocket and editing pocket which has important role in proofreading.
Catalytic pocket of each aaRS has specificity for their respective amino acid. For example in case of valine and Isoleucine. The valine aaRS can easily discriminate between valine and Isoleucine due to large size of Isoleucine, valine easily slip into Isoleucine catalytic pocket but excluded because methylene group of Isoleucine provide extra free energy which 100 fold increase the binding of Isoleucine then val. Use in proofreading during adenylation reaction.
Editing pocket present in deep cleft of enzyme and greatly increase the proofreading. Now LET'S TALK some example of catalytic pocket, AMP-valine easily come in to editing pocket of IIe and get hydrolyzed into valine and AMP. But Isoleucine-AMP. not able to enter in its editing pocket and remain binded, thus the binding again increase by 100 fold, in combination of both pocket proofreading mechanism it increase up to 10000 fold that is 0.01 %.
5.10.2 At the level of Ribosome: Three mechanism are there to increase the fidelity of codon- anticodon pairing.
Kinetic proofreading–Once tRNA get charge with amino acid then binds by EF-Tu, which bring charge tRNA to translation apparatus and facilitate correct codon and anti codon pairing but restrict the incorporation of amino acid in polypeptide chain, as correct pairing occur between codon and anti codon same time hydrolysis of EF-Tu bind GTP occur. GTPase activity of EF-Tu very sensitive towards correct pairing. Incorrect codon-anticodon pairing at A site of ribosome greatly reduce the activity of GTP hydrolysis. This allows the dissociation of EF-Tu with incorrect tRNA from ribosome.
EF-Tu create short delay period between correct pairing and incorporation of amino acid, in between this time duration only cognate tRNA remain bind due to strong hydrogen bonding and noncognate tRNA release due to weak hydrogen bonding. As a result only correct amino acid incorporate in growing chain.
Two Adenine residue with in 16 S rRNA sequence make tight interaction with minor grove of correctly paired anticodon and first two base of codon, as we know that in normal Watson-Crick pairing no difference notices in minor grove of correctly pair base, but in case of Non Watson-Crick pairing difference occur and get dissociate by ribosome because not any kind of interaction occur with A residue.
After the release of EF-Tu, 3’ end of tRNA present at A site not in a proper position to make peptide bond need to rotate in peptidyl center of large subunit, according to this only correctly paired tRNA or cognate tRNA bear the strain create by the rotation otherwise noncognate tRNA get dissociated.
Some topic related to translation
After the partial synthesis of a protein some ribosomes that are stalled on mRNA prior to it reaches a stop codon may be untied by the action of tmRNA, because they require releasing factor to get release from mRNA and release factors will not load without stop codon, which provide by tmRNA. tmRNA include structure of both tRNA and mRNA. tmRNA contain 12 helices (also known as P1 to P12). The tRNA like region in tmRNA possess 5' monophosphate and 3' CCA ends and tRNA-like domain (TLD). TLD contain helices 1 analogue of tRNA acceptor stem, helices 12 analogue of T-stem and helices 2 analogue of variable stem. mRNA like region which is possess by tmRNA called as mRNA-like region (MLR).
MLR is like a large loop having a coding sequence (CDS) marked by resume codon and the stop codon for the tag peptide, In short, tmRNA encodes a short open reading frame (ORF) of about 10 aa with stop codon in its MLR region for tag peptide, which bring this protein for degradation by protease. If a ribosome is stalled in between mRNA then alanine charge tmRNA enter the A site of ribosome and alanine is added to the emergent polypeptide chain, and then the ribosome shifts from the mRNA to the tmRNA open reading frame (ORF). Release factor binds to the tmRNA stop codon to stop translation and release the ribosome after the 10 amino acid tmRNA-encoded peptide is added and this tmRNA-encoded peptide tags the protein for degradation by a protease. MLR region also contain four pseudoknots, which are conserve. One pseudoknots present upstream of CDS and three pseudoknots downstream of CDS. In bacteria, depending on the set of proteases and adaptors available various type of tag peptide present, in E.coli present as a tag peptide.
5.12. Translational Bypassing: The process of resuming the translation by ribosome some of 50 nucleotides downstream of stop codon after translational stop at stop codon and get release from mRNA.
5.13. Coupled translation
When translation of a downstream cistron is coupled to that of the preceding cistron in polycistronic transcripts called as coupled transcription. Coupled translation occur because of following reason.
- If functional RBS (Ribosome binding site) of downstream cistron temporarily obscured by secondary structure.
- If downstream cistron lack a capable RBS that’s why ribosomes are convey to the downstream cistron upon completing translation of the preceding cistron so it involves retention and reuse of ribosomes but this occur only in one condition when the terminator codon of one gene often extend beyond the start codon of the next (e.g.) and this proximity assist reinitiation of translation.
A number of advantage of coupled transcription, In E. coli foreign gene expression can be increase by copying this arrangement. Coupled translation is also employ to manage gene expression–e.g. allowing production of several ribosomal proteins to be turned on or off via a single control point in the mRNA–but coupling does not essentially guarantee the same concentration protein yields. Coupled translation has the unpredicted benefit of increasing folding of the protein derived from the downstream cistron.
Rarely, translation is obligatory for transcription to carry on. It is the case of Rho-dependent polarity results when the lacks of translation of an upstream open reading frame permit rho to bind and terminate transcription prematurely.
5.14. Suppressor tRNAs
A suppressor tRNA able to changes the codons to which it responds because it has mutation in their anticodon. Thus suppressor tRNA able to read stop codon because its anticodon not read stop codon as stop codon instead of read as any codon of normal amino acid. So as emergent polypeptide chain able to extend by addition of amino acid beyond the termination codon because new anticodon corresponds to a termination codon. In some rare suppressor tRNAs mutation is reported other part of molecule. There is competition occur between suppressor tRNAs and wild-type tRNAs because both have same anticodon to read the corresponding codon(s). Thus competent suppression is harmful because it results in readthrough past normal termination codons. For example in UGA codon, which is leaky, thus misread by Trp-tRNA at 1% to 3% frequency.
There are some mutational type effect that get suppress by suppressor tRNA.
Suppressor tRNA cause nonsense suppression at a site of nonsense mutation, or in readthrough at a usual termination codon through its mutant anticodon. By mutated anticodon of suppressor tRNAs each type of nonsense codon mutation is suppressed
Missense suppression occurs when tRNA recognizes a different codon form due to missense mutation and add normal one which is previously present before missense mutation occur, so that one amino acid is substituted for another.
Frameshifting also occur during translation due to some slippery sequences and downstream RNA structure (pseudoknot and stem loop but not all pseudoknot and stem loop). This is also known as translational framshifting. The sequence of mRNA and the ribosomal environment responsible for shift in reading frame of mRNA in which it coded. Changing in the reading frame occur when slippery sequences, which is typically a heptanucleotide (XXXYYYZ, where X=G, A and U, Y= U and A and Z = C, A and U) allow a tRNA to shift by one base after it has paired with its anticodon. If ribosome shift one base in 5’ direction it represent as -1 and if ribosome shift in 3’ direction it represent as +1, these are known as classes of signal that represent direction of shifting of ribosome’s movement. Translational fusion of the two overlapping open reading frames also result from translational framshifting, thus the information for the formation of protein comes from two distinct open reading frame and the protein from both of them have common N terminal.
Some genes translation depends upon the usual occurrence of programmed frameshifting. It is mainly observe in case of viruses, which uses this mechanism to produce correct proportion of protein for their maturation and particle assembly. This is first observed in virus, eg. retrovirus Rous Sarcoma Virus (RSV) for protein is produced by gag and pol ORFs. In retrovirus -1 translational framshifting cause the production of 95% Gag protein and 5% Gag-pol protein.
Recording : When the sense of a codon or series of codons is changed from that expect by the genetic code this type of event called recording. Due to ribosome interactions between aminoacyl-tRNA and mRNA get altered.
5.16. Control of Initiation
Prokaryotic Translational Control: Translation efficiency depends on the structure of mRNA, as mRNA as translate when it is not present in secondary structure, if initiation codon present with in secondary structure then it effects the translational initiation rate as if initiation codon present with in some other gene cistron then also rate of translation initiation effect because depends on the translation of the gene in which initiation codon buried and rate of translation initiation also affected if it is the case of coupled translation.
For example, in MS2 family of RNA phages initiation codon of replicase cistron present within double-stranded structure that also engage the part of the coat gene, replicase so translation of replicase gene not occur until the translation of coat protein occur because during the translation of coat protein ribosome unwinds the secondary structure in which initiation codon of replicase gene hides. Feedback repression also controlled Prokaryotic translation. Another example from one ribosomal protein genes mRNA comprise cistrons encoding both the L11 and L1 ribosomal proteins, both of them having couple translation in which L11 encoded previously then L1.When L1 protein present in moderate amount, binds firmly to a hairpin loop structure in 23S rRNA which is analogous to the stem loop structure present in close proximity to the translation start site of the L11 cistron. When L1 (and L11) are more plentyful than 23S rRNA, L1 binds to a similar stem loop structure present in close proximity to the translation start site of the L11 cistron. Thus translation of both L11 and L1 cistrons repress due to coupled translation.
5.17. Eukaryotic Translational Control
Eukaryotic mRNAs has longer life span than prokaryotic ones, so there is more chance for translational control. Initiation is rate-limiting factor in translation, thus most control apply at this stage. Generally the phosphorylation of initiation factors involve in control mechanism of initiation and this phosphorylation either stimulatory or inhibitory. For example, a protein binds directly to the 5’ untranslated region of an mRNA and inhibits its translation; exclusion of this protein activates translation.
5.17.1. Phosphorylation of Initiation Factor eIF2α
It is an example of inhibitory phosphorylation take place in reticulocytes, which make one protein haemoglobin. When reticulocytes are starved for heme, then formation of α-globins and β-globins seems wasteful. Deficiency of heme unmasks the activity of heme controlled repressor (HCR) which is a protein kinase and phosphorylate eIF2α, a subunit of trimeric eIF2. The phosphorylated eIF2 binds more strongly than normal to eIF2B. The elF-2B is GEF and also an initiation factor and block eIF2B to perform its function. thus other eIF2 remains in the inactive GDP-bound form and cannot attach Met-tRNAi Met to 40S ribosomes. Thus, responsible for translation initiation halt.
Another example comes from Interferons which is an antiviral protein. At the time of viral infection, due to the presence of interferon and doublestranded RNA a double-stranded RNA-activated inhibitor (DAI) a eIF2α kinase activated mechanism same like HCR to block the translation of virus in virus infected cell. Thus eIF2α phosphorylation condition unfavorable for cell growth, which shown in both above mention cases.
Phosphorylation of an eIF4E- cap binding protein which stimulates translation initiation. Phosphorylated eIF4E binds the cap with about four times more affinity then unphosphorylated eIF4E, thus cause stimulation of translation and favorable for cell growth. Cell division stimulation with insulin or mitogens cause increase in eIF4E phosphorylation and platelet derived growth factor (PDGF) also stimulate translation in mammals by an another pathway that occupy eIF4E.
Insulin binds to insulin receptor, present on cell surface. Insulin receptor is a receptor tyrosine kinase which activates a protein called mTOR. A protein called PHAS-I is a target of mTOR. PHAS-1 inhibit eIF4E activity.
PHAS-1 binds with elF4E. But once mTOR phosphorylate PHAS-I (2) It no longer can bind with elF-4E. Thus elF-4E is free and can participate in translation.
5.18. mRNA surveillance
mRNA surveillance mechanisms are pathways utilized by organisms to ensure fidelity and quality of messenger RNA (mRNA) molecules. There are a number of surveillance mechanisms present within cells.
mRNA surveillance is an enigmatic process because it requires a cellular machinery that can discriminate normal from aberrant mRNAs. mRNA surveillance has been documented in bacteria and yeast. In eukaryotes, these mechanisms are known to function in both the nucleus and cytoplasm.
Biology of mRNA turnover
The steady-state level of a given mRNA depends on the balance between its rates of synthesis and degradation. Importantly, the decay rate of mRNA can be changed to control the amount of polypeptide the cell produces.
The regular mRNA is decay either by decapping followed by 5’-3’ exonuclease or/ and by deadenylation followed by 3’-5’ exonuclease. There is one more mechanism exist to decay the mRNA that is degradation of mRNA by endonulcease followed by 5’-3’ exonuclease and 3’-5’ exonuclease.
Three surveillance mechanisms are currently known to function within cells:
- the nonsense-mediated mRNA decay pathway (NMD);
- the Nonstop Mediated mRNA decay pathways (NSD);
- the No-go Mediated mRNA decay pathway (NGD).
Deadenylation is the most common route of mRNA degradation followed by decapping and 5'® 3' exonucleolytic decay.
5.18.1. Nonsense-mediated mRNA decay (NMD)
The NMD pathway acts via deadenylation-independent decapping, followed by 5'® 3' exonucleolytic decay whereas nonstop decay appears to proceed via deadenylation-independent 3' ® 5' exonucleolytic decay. In bypassing the rate-limiting step of deadenylation, the mRNA surveillance pathways allow the rapid removal of irregular mRNAs from Nonsense-mediated decay (NMD). Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that exists in all eukaryotes.
NMD reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. Three interacting trans-acting factors, Upf1p, Upf2p, and Upf3p, are required for NMD but play no role in nonstop decay. Following splicing in the nucleus, the exon junction complex (EJC), which contains UPF3 (a core protein of the NMD pathway), is associated with the transcript, and the resulting messenger ribonucleoprotein is exported out of nucleus to the cytoplasm.
All three of these factors are trans-acting elements called up-frameshift (UPF) proteins. In mammals, UPF2 and UPF3 are part of the Exon-exon Junction complex (EJC). exon-exon junction is formed after splicing. UPF2 and UPF3 bound to mRNA other proteins, eIF4AIII, MLN51, and the Y14/MAGOH heterodimer, which also function in NMD. UPF1 phosphorylation is controlled by the proteins SMG.
In NMD eRF1, eRF3, Upf1, Upf2 and Upf3 make the surveillance complex. These protein scans the mRNA for premature stop codons. The assembly of this complex is triggered by premature translation termination. Premature termination codon can arise at the DNA level by mutations or at the level of RNA by transcription errors or alternative pre-mRNA splicing. If a premature stop codon is detected then the mRNA transcript is signaled for degradation – the coupling of detection with degradation occurs. Normally the premature stop codon are found 50-55 nucleotide upstream of exon exon junction.
In normal mRNA the Exon-exon Junction Complex (EJC) is upstream to stop codon. The Exon Junction complex get dissociated by the ribosome during the first round of translation. However the premature translation termination found upstream of exon exon junction. This implies that the Exon Junction complex protein remain bound to the mRNA even after this first round of translation, as the ribosome get dissociated before the exon exon junction. This activates the NMD. The premature termination of translation leads to the assembly of a complex composed of UPF1, SMG1 and the release factors, eRF1 and eRF2, on the mRNA.
If an exon Junction complex is left on the mRNA because the transcript contains a premature stop codon, then UPF1 comes into contact with UPF2 and UPF3, triggering the phosphorylation of UPF1. Phosphorylation of UPF1 by SMG1 leads to dissociation of eRF1 and eRF3 and binding of the SMG7 adaptor protein. If the premature translation termination within about 50 nucleotides of the final exon-junction complex then the transcript is translated normally. However, if the termination codon is further than about 50 nucleotides upstream of any exon-junction complexes, then the transcript is down regulated by NMD.
The phosphorylated UPF1 then interacts with SMG-5, SMG-6 and SMG-7, which promote the dephosphorylation of UPF1. SMG-7 is the most important protein for NMD thus called as terminating effector in NMD. SMG-7 also accumulates in P-bodies, which are cytoplasmic sites for mRNA decay. In both yeast and human cells, the major pathway for mRNA decay is initiated by the removal of the 5’ cap followed by degradation by XRN1, an exoribonuclease enzyme. The other pathway by which mRNA is degraded is by deadenylation from 3’-5'.
In the cytoplasm, a second NMD core protein, UPF2, binds to UPF3. Ribosomes associate and translate the mRNA, but are stalled on encountering a premature termination codon (PTC). This results in binding of the SURF complex (comprising SMG1, UPF1 and the peptide-release factors eRF1 and eRF3) to the ribosome. UPF1 also binds UPF2, thereby linking the EJC to the PTC. Subsequent steps that are still being elucidated lead to mRNA decay by various pathways.
The second method for degradation of mRNA is Non Stop decay.
5.18.2. Non-stop decay.
Non stop mRNA lacks stop codon i.e the mutation in DNA create a condition in which the stop codon of mRNA is converted into a sense codon and allow translation to continue. Translation of a mRNA which lacks a stop codon results in ribosomes traversing the poly(A) tail, displacing poly(A)-binding protein (PABP) and stalling at the 3' end of the mRNA. To release the ribosome form mRNA releasing factor binds with stop codon and later ribosome recycling factor dissociates the ribosome form mRNA.
In yeast and mammalian cells, Ski7 play a role in non stop decay. Ski7 is an adaptor protein that functions as a molecular mimic of tRNA, binds to the A site on the stalled ribosome to release the transcript, and then recruits the exosome. The exosome degrades the poly(A) tail and later the complete mRNA .
In another pathway described in Saccharomyces cerevisiae, in the absence of Ski7, the displacement of PABP by the translating ribosome renders the mRNA susceptible to decapping and 5' 3' decay by the 5' 3' exoribonuclease Xrn1.
5.18.3. An another mechanism for decay of mRNA
Several time because of the strong secondary RNA structure formation within the open reading frame (ORF) the ribosomes stall on the mRNA. That means the ribosome is not able to move on the mRNA thus called as No-go decay.
The Dom34 and Hbs1 proteins bind the transcript near the stalled ribosome and initiate an endonucleolytic cleavage event near the stall site. This releases the ribosome and generates two mRNA fragments, each with a free end exposed for exonucleolytic decay by the exosome and Xrn1, respectively.