5.5. Aminoacyl tRNA synthetases (aaRS)
Aminoacyl tRNA synthetases (aaRS) is responsible for the attachment of amino acid with tRNA. Aminoacyl tRNA synthetases are specific for attachment of each amino acid with its cognate tRNA.
aaRS are two type:
- Class I aaRS attach amino acid to 2’ OH of terminal nucleotide. Which is adenine. Class I aminoacyl tRNA synthetases are monomeric enzyme. Class I enzymes contain HIGH and KMSKS motifs called as Rossmann nucleotide fold. Class I responsible for charging of Arg, Cys, Gln, Glu,Ile, Leu, Met, Trp, Tyr, Val.
- Class II Aminoacyl tRNA synthetases attach amino acid to 3’ OH of adenine neucleotide. Mostly in dimeric proteins that reaction found. Class II enzymes have an unrelated β-sheet arrangement and are describing by three degenerate sequence motifs. Class II responsible for charging of Ala, Asn, Asp, Gly, His, Lys, Phe, Pro, Ser, Thr.
Aminoacyl tRNA synthetases (aaRS) use specific mechanism to identify cognate tRNA by recognize accepter arm and anticodon loop. Accepter arm also called as discriminator arm, because single base pair (this base is known as discriminator base) change in accepter arm change the specificity of aaRS towards that tRNA. Thus the tRNA determinants (group of bases or base) use by aaRS to discriminate between cognate and non cognate are called second genetic code. They are testred as second genetic code because play crucial role in information flow from mRNA to protein.
5.6. Steps of Translation In Prokaryoties
- Activation and charging of amino acid
5.6.1. Activation and charging of amino acid :- The activation and changing is a two step process.
The Aminoacyl tRNA synthetases catalyze activation of amino acid and charging of amino acid to tRNA with the help of ATP. Both reactions carried out with in active site of enzyme.
Step 1: Activation of the Amino Acid, with the involvement of ATP. The amino acid present in cell are inert molecule and need to be activate by ATP.
Amino acid (aa) + ATP <=> aa~AMP (aminoacyl-adenylylate intermediate) + PPi
Step 2: Transfer of the aminoacyl group to the tRNA, Aminoacyl groups attached to the 2' hydroxyl or 3' hydroxyl group of the terminal ribose by Class I enzymes and Class II enzyme respectively. This reaction is called as aminoacylation. Amino acid attached to tRNA by high energy bond. In this reaction one O (oxygen) is removed from amino acid.
Three initiation factor IF1, IF2, and IF3 takes part in initiation. 70S ribosome get dissociated into 50S and 30S subunits, under the influence of IF-1. IF-1 binds to A site and prevents an aminoacyl-tRNA from entering. IF3 dissociates the 70S-ribosome and thus suppling the pool of free 30S subonits required for translation initiation.
Along with AUG, GUG or UUG also act as initiation codon and three of them always code for methionine if act as initiation codon.
An interior AUG always code for methionine, if act as initiation codon or present internally with in polypeptide, but GUG and UUG code for valine and leucine, respectively if present internally within polypeptide. UUG act as initiation codon in gram-positive bacteria and some bacteriophage. Translation is less efficient when an alternative start codon replaces AUG due to weaker pairing (two rather than three base pairs) with fMet tRNA.
Formation of 30S initiation complex complete as three initiation factors along with initiator tRNA binds to mRNA. Due to the pairing between codon and anti codon then the small subunit undergo conformational change and release of IF3 occur.
After that 50S subunit joins, as a result hydrolysis of IF2 bound GTP to GDP and Pi by factor binding center or GTPase center present in 50S subunit at A site cause release of IF2 and IF1. Now formations of 70S complete.
After 70S ribosome formation translation proceeds to elongation phase. Elongation start with the binding of amino acyl tRNA to A site. This is mediated by elongation factor EF-Tu. EF-Tu is a G protein and active when binds to GTP. Now EF-Tu GTP transfer amino acyl tRNA to A site. EF-Tu generally present in complex with EF-Ts.
When EF-Tu binds to GTP become active and able to binds with amino acyl tRNA to its 3’ end and masking the charge amino acid. This ternary complex (aminoacyl-tRNA EF-Tu-GTP) move towards A site because the P site is already occupied by initiator tRNA. EF-Tu binds in similar manner like IF2 and in two step aminoacyl-tRNA is loaded into the A site. EF-Tu bind non specifically with all charge tRNA.
Aminoacyl-tRNA is loaded into the A site in two steps process. First, the anticodon loop binds to this change in conformation the 30S subunit’s A site and second, codon-anticodon recognition and pairing mediate conformational change in ribosome. Stabilizes tRNA binding and cause EF-Tu to hydrolyze its GTP. After hydrolysis binary complex EF-Tu-GDP is released and binds to EF-Ts. EF-Ts is a G nucleotide exchange factor (GEF) covert EF-Tu- GDP into EF-Tu-GTP and this activated EF-Tu binds to new amino acyl tRNA. This process is known as decoding.
Now the first peptide bond is formed between peptidyl tRNA and initiator tRNA. Amino group of amino acid of initiator tRNA attack the carbonyl group of amino acid which linked to peptidyl tRNA by ester linkage, result in the formation of tetrahedral intermediate and this complex resolve to form peptide bond with the release of deacylated tRNA.
The peptide bond is formed by peptidal transferase enzyme present in large subunit and the main catalysis perform by 23 S rRNA having ribozyme activity hence peptidal transferase activity present in 23S rRNA. This is proof by high resolution structure of ribosomal large subunit. It reveal that no amino acid of protein is positioned at the active site where the transfer reaction between peptidyl-tRNA and aminoacyl-tRNA takes place. From this study it reveal that sugar of adenine residue of RNA perform this reaction. In this process of peptide bond formation one H+ release by second amino acid.
After that ribosome move forwards three nucleotides along the mRNA. This is known as ribosomal translocation. Translocation causes deacetylated tRNA to transfer from the P site to the E site and A site tRNA from A site to P site. The A site is free for new amino acyl tRNA.
The translocation is carried out by EF-G. EF-G is a G-Proteins. It binds with GTP. As a result EF-G-GTP bind to A site near factor binding centre and interact with them, because its binding site get free due to sift in A site tRNA. Than GTP of EF-G- GTP get hydrolysed and convert into EF-G- GDP. The GTP hydrolysis release energy it moves to small subuite and mediate the transfer of anticodon loop of A site tRNA to P site. Translocation get complete as result EF-G-GDP affinity also get reduced and it leaves the ribosome. The 50S moves relative to the 30S and then the 30S moves along m RNA to restore the original conformation. Rate of translation is 15 amino acid (45 nucleotide) per second.
The formyl residue on the initiator methionine is removed from the protein by a specific deformylase enzyme to generate a normal NH2 terminus within bacteria and mitochondria. If methionine is to be the N -terminal amino acid of the protein, this is the only necessary step. About half the polypeptides in which methionine at the terminus is removed by an aminopeptidase, which creates a new terminus from R2 (second amino acid incorporated into the chain). When both steps are necessary, they occur sequentially. The removal reaction (s) occur rather rapidly, when the nascent polypeptide chain has reached a length of 15 amino acids.
5.6.4. Termination :
The termination of translation occurs when a stop codon comes at ribosomal A site and recognize by class 1 release factors (RF1). Which decode stop codon UAA and UAG and RF2 which decode stop codon UAA and UGA when presented in the A site. Releasing factors utilize a localized linear amino acid sequence for the identification of a stop codon because RF1 and RF2 posses overlapping codon specificity. The switch of RF1- and RF2-specific domains between RF1/RF2 chimeric factors led to the recognition of a anticodon within each factor. The sequences RF1 contain PAT and RF2 contain SPF sequence were capable of arresting either RF1 or RF2 precisely on the chimeric factor as per requirement of RF1 or RF2.
RF I or RF2 consists of three domains that mimic the structure of tRNA. The second domain has three amino acids motif known as GGQ Glutamate, Glutamate, Glutamine. The GGQ is also called peptidyl anticodon. The exposed GGQ represent amino acid which is charge by amino acyl-tRNA present at A site and its Q use to present an H2O as a substitute of amino acid within peptidyl transfer reaction and the transfer of OH from H2O to the peptidyl-tRNA hydrolysed the peptidyl-tRNA linkage and OH attached to peptidyl tRNA and O move to polypeptide, as a result polypeptide get release but RF1 or RF2, deacylated tRNA and the mRNA remain present on ribosome.
Class 2 factor RF3 (related to EF-G) responsible for the release of RFI or RF2. RF3 is a G protein but has more affinity toward GDP in presence of class I release factor. Thus RF3-GDP binds to the ribosome before the termination reaction occurs but as the polypeptide release cause the conformational change in ribosome and class I release factor mediate replacement of GDP in to GTP. This exchange facilitate RF3 to mediate high affinity interaction with ribosome in turn release of class I release factor occur. Now RF-3 interacts with ribosome GTPase centre, result in GTP hydrolysis. The RF-3 is converted into RF3-GDP form, which has less affinity for ribosome in absence of class I release factor thus cause release of RF-3.
Recycling factor (RRF) which mimic tRNA but lacks 3’ structure facilitate dissociation of the remaining components tRNA, mRNA 30S, and 50S subunits in cooperation with EF-G and IF3. EF-G mediated translocation of uncharged tRNA from P and E sites same like in elongation by displacement of RRF from A site uses hydrolysis of GTP. RRF operate on the 50S subunit and deacylated tRNA from the 30S subunit remove by IF-3 as well as IF3 participate in release of mRNA require for separation of both subunit and as subunit get separate then IF3 remain bound with small subunit necessary to prevent their reassociation.
5.7. Steps of translation in Eukaryotic
- Termination Activation and charging of amino acid
5.7.1. Activation and charging of amino acid
Its mechanism is same like prokaryote in spite of one difference, here initiator methionine amino acid not formylated and its charge mRNA called tRNAi Met.
In eukaryotes Ribosome binding site is absent. Initiation starts when the cap at the 5’-end recognize by initiation factor bind to small ribosomal subunit 40S and making 43S complex, which direct initiate others factors to bind with ribosome and scanning the mRNA in the 5’→3’ direction until they encounter start codon. The start codon is present with in Kozak sequence (5’CCRCCAUGG3’ where R is a purine – A or G).
80S ribosome dissociate in 40 S and 60S by eIF6, which binds to the 60S ribosomal subunit and then eIF1A and eIF3 analogue of prokaryotic IF3 binds to small subunit 40S, all of them prevent premature association of 60S with the 40S subunit. After that eIF2, which is a trimeric G protein binds to GTP and become active and now binds to tRNAi Met form ternary complex eIF2 - GTP- tRNAi Met. eIF5A stimulates the GTPase activity of eIF2 and binds to b-subunit of eIF2. This complex bind to 40S and this binding is facilitated by eIF1A and eIF3 and form 43S complex.
Another initiation factor, eIF4F is a hetrotrimer, complex of three subunit binds to mRNA. eIF4F contain eIF4E which binds to 5' cap of mRNA. The eIF4G a scaffold protein binds to eIF4E and PABP (poly A binding protein). This cause circularization of mRNA and eIF4A has helicase activity. The eIF4A causes unwinding of 5’ cap of mRNA and be with mRNA during the whole elongation process. eIF4A activity depends on ATP, simultaneously eIF4B which has RNA-binding domain join the complex and stimulate eIF4A helicase activity.
The eIF4F loads the mRNA onto the 43S complex by making interaction with eIF3 by central portion of eIF4G and the poly(A) binding protein (PAB). Tethering among eIF4E and eIF3, eIF4G can bring the 5’-end of the mRNA in close proximity to 43S particle, thus 43S begin scanning in association with eIF1 and eIF1A, which play important part in this scanning process. eIF1 also enhance eIF3 dissociation activity. This whole complex called 48 S complex.
The scanning stop when eIF5 recognise and binds CCRCC of kozak sequence. The base pairing occurs between the initiation codon and the initiator tRNA. This facilitate GTP hydrolysis by eIF2. GTP hydrolysis is mediated by eIF5. It has GTPase activating protein (GAP) activity. After GTP hydrolysis, eIF2-GDP liberate from P site of ribosome and the Met-tRNAi remain at the P site. The eIF2-GDP get converted in to eIF2-GTP by eIF2B has Ghucleotide exchange factor (GEF).
This trigger binding of eIF5B-GTP to the complex which assists the joining of the large (60S) ribosomal subunit to the 40S Met-tRNAi- mRNA complex. This mediate GTP hydrolysis by eIF5B, and eIF5B-GDP form, it has low affinity for the ribosome, thus release from the complex. eIF5B has similar sequence to prokaryote. When the complete 80S ribosome is formed after that other factor (eIF3, eIFIA, eIF5) also release and end of translation initiation occur.
It's mechanism is same like prokaryote in spite change in name of factor for respective function. So here LET'S TALK about the mentioned name of factor and its respective analogue in prokaryotes, both having same function.
EF-Tu = eIF1α
EF-Ts = eIF1β / γ (GEF)
EF-Tu-EF-Ts = eIF1α- eIF1β / γ
EF-G = eEF2
Rate of translation in eukaryote 2-4 amino acids per second. In eucaryotes translation is not coupled to transcription. New amino acid attached to C terminal of growing polypeptide chain and N terminal contain first amino acid attached to polypeptide chain.
It's mechanism is same like prokaryote in spite change in name of factor for respective function. So here LET'S TALK about the mentioned name of factor and its respective analogue in prokaryotes, both having same function.
Class I release factor (RF I and RF II) = Class I release factor RF I (only one release factor present code for all three stop codon). And the recycle of ribosome is achieved by eukaryotic ribosome recycling factor.
The sequence of dissociation and association of ribosome during translation called ribosome cycle. Each mRNA can translate by multiple ribosome simultaneously that type of ribosome called polysome or poly ribosome. A ribosome covered 80 nucleotide on mRNA due to large size but make interaction of mRNA with only
30-35 nucleotide thus this polyribosome exist, this show limited concentration of mRNA in cell.
Some protein contain selenocystein, which is coded by UGA. This UGA able to encode selenocystein due to the presence of special sequence known as SECIS element within mRNA of those protein. In eukaryotes and archaea it is located in 3’ UTR and can express multiple UGA codons to encode selenocysteine residue. In prokaryotes it is located just after UGA codon within the ORF of respective protein. The selenocysteine tRNAs (tRNASeC) are initially charged with serine. This Ser-tRNASeC is converted to a selenocysteine residue by selenocysteine synthase and recognize by selenocystein specific elongation factor SelB in prokaryote and mSelB or eEFSec in eukaryote.
Those elongation factors bring Sec-tRNASec to ribosome by recognition of SECIS element and by interacting with its RNA secondary structures which is created by the SECIS elements in selenoprotein mRNAs. Sec-tRNASec, having some special feature like long variable arm, replacement at several well-conserved base positions and its accepter arm in prokaryote having 8 and in eukaryote 10 base pair.
Unusual mRNA structures and alternative initiation mechanisms
In leaderless mRNA AUG codon place precisely at the 5′ end might be capable to enter into the groove between the 30S and 50S subunits. Leaderless mRNA interact more firmly to 70S ribosomes than to the 30S. IF3 inhibited translation of leaderless mRNAs. Which support dissociation of 70S ribosomes, and amplified by IF2, which maintain binding of fMet-tRNA. this mechanism alternative to the standard sine delgerno (SD) mediated initiation mechanism. In another method, Epsilon sequence present upstream to sine delgerno (SD) sequence and govern pairing between mRNA and 16S rRNA or a sequence positioned downstream to AUG.
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.