Recognition Of Initiation Codon Research Articles

Ribosome assembly defects subvert initiation Factor3 mediated scrutiny of bona fide start signal.

In bacteria, the assembly factors tightly orchestrate the maturation of ribosomes whose competency for protein synthesis is validated by translation machinery at various stages of translation cycle. However, what transpires to the quality control measures when the ribosomes are produced with assembly defects remains enigmatic. In Escherichia coli, we show that 30S ribosomes that harbour assembly defects due to the lack of assembly factors such as RbfA and KsgA display suboptimal initiation codon recognition and bypass the critical codon–anticodon proofreading steps during translation initiation. These premature ribosomes on entering the translation cycle compromise the fidelity of decoding that gives rise to errors during initiation and elongation. We show that the assembly defects compromise the binding of initiation factor 3 (IF3), which in turn appears to license the rapid transition of 30S (pre) initiation complex to 70S initiation complex by tempering the validation of codon–anticodon interaction during translation initiation. This suggests that the premature ribosomes harbouring the assembly defects subvert the IF3 mediated proofreading of cognate initiation codon to enter the translation cycle.

Polyamines release the let-7b-mediated suppression of initiation codon recognition during the protein synthesis of EXT2.

Proteoglycans (PGs), a family of glycosaminoglycan (GAG)-protein glycoconjugates, contribute to animal physiology through interactions between their glycan chains and growth factors, chemokines and adhesion molecules. However, it remains unclear how GAG structures are changed during the aging process. Here, we found that polyamine levels are correlated with the expression level of heparan sulfate (HS) in human skin. In cultured cell lines, the EXT1 and EXT2 enzymes, initiating HS biosynthesis, were stimulated at the translational level by polyamines. Interestingly, the initiation codon recognition by 43S preinitiation complex during EXT2 translation is suppressed by let-7b, a member of the let-7 microRNA family, through binding at the N-terminal amino acid coding sequence in EXT2 mRNA. Let-7b-mediated suppression of initiation codon depends on the length of 5′-UTR of EXT2 mRNA and its suppression is inhibited in the presence of polyamines. These findings provide new insights into the HS biosynthesis related to miRNA and polyamines.

EIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5′-end of messenger RNA (mRNA) and scans its 5′-untranslated region (5′-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons—following structured 5′-UTRs, in bad AUG context, within few nucleotides from 5′-end of mRNA and CUG start codon—is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.

English

Introduction Organisms are able to generate organised responses to environmental changes by controlling the level of messenger RNA translation. The ability to respond rapidly allows the mobilization of the host’s defences and coordination of innate response to different infections. A large number of microbial pathogens inhibit the translation of host cells. Bacterial and virus infections interfere with protein synthesis of the host and activating or suppressing the innate responses, thus manipulating the translational machinery. Mortality, morbidity and economic adverse effects associated with fungal infections along with the emergence of resistance to antifungal agents make it necessary to gain a better understanding of the pathogenesis and discovery of new agents for treatment of those infections. There is little knowledge regarding the role of translational regulation in fungus–host relationship. The present review aims to show how translation regulation occurs in bacteria and virus infections and shows a mysterious role of translational regulation in the context of fungal infections. Conclusion In the last few years, incidence of fungal diseases has dramatically increased especially in immunodepressed individuals, representing a serious health problem since these diseases are associated with high mortality. There is a growing list of reports describing antifungal resistance in clinical isolates. Considering these facts, it is necessary for a better understanding of the pathophysiology of fungal infections that may reveal new therapeutic targets. Introduction The regulated translation of mRNAs in eukaryotes is a powerful tool of posttranscriptional control of gene expression. Translation has a fundamental role in cell growth and differentiation, development, learning, memory and synaptic plasticity. This process and its regulation have a relevant role in infections and host defence, allowing quick responses to contain pathogenic microorganisms. Regulation of these events occurs primarily at the steps of initiation and elongation. Figure 1 resumes the process. Before mRNA decoding, the eukaryotic 40S ribosomal subunit forms a 43S pre-initiation complex composed of different eukaryotic translation initiation factors (eIFs) and initiator tRNA charged with methionine (Met-tRNAi—see Figure 1). This association involves elF2, a heterotrimer that forms an active ternary complex (TC) containing GTP and aminoacylated initiator tRNA. In the presence of eIF4F complex, TC carries Met-tRNAi to 40S ribosome, a fundamental step to start protein synthesis at AUG codon or cognates1. eIF4F binds to the cap structure present in the 5 -end of eukaryotic mRNAs. This interaction promotes correct positioning of ribosome relative to mRNA, thus facilitating the ATP-dependent movement of it that is named as ‘scanning’ and locates the translation initiation codon AUG2. eIF4F is composed of a cap-binding protein (eIF4E), an RNA helicase (eIF4A) and a scavenger protein (eIF4G). eIF4E binds to the cap while eIF4G associates with eIF3 linked to a ribosome, allowing correct positioning of 43S complex at the mRNA 5 -end. Besides, eIF4E binds to PABP (poly(A)-binding protein) connecting 5 and 3 -ends of mRNAs, resulting in mRNA circularization that is supposed to be important for a rapid turnover in protein synthesis1. Translation initiation is the limiting step of protein synthesis in most circumstances, and then the process is subject to elegant regulatory mechanisms1. Initiation step is regulated by the availability of eIF4F2. The binding of eIF4E to eIF4G is modulated by a serine/threonine kinase, TOR1, which responds to growth factors, nutrients, amino acids, oxygen and energy availability3. In this context, TOR1 in a TORC1 complex, which controls eIF4E activity through phosphorylation of 4E-BPs (eIF4Ebinding proteins). Hyperphosphorylated 4E-BPs interact with eIF4E, preventing the formation of eIF4F complex and thus suppressing the cap-dependent translation, while hypophosphorylation of these proteins by mTORC1 promotes the release of eIF4E-eIF4G binding, stimulating the cap-dependent translation. After AUG initiation codon recognition, eIF5 stimulates the hydrolysis of GTP bound to eIF21. In a reaction catalysed by eIF5B, the 40S ribosomal subunit binds to 60S and * Corresponding author Email: gouveia_va@yahoo.com; gouveiava@ ufmg.br Laboratorio de Biologia Celular de Microrganismos, Departamento de Microbiologia – ICB, Universidade Federal de Minas Gerais, Brazil

Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation

Start codon recognition is a crucial event in the initiation of protein synthesis. To gain insight into the mechanism of start codon recognition in eukaryotes, we used a yeast reconstituted initiation system to isolate the step of Met-tRNA(i)*eIF2*GTP ternary complex (TC) binding to the 40S subunit. We examined the kinetics and thermodynamics of this step in the presence of base changes in the mRNA start codon and initiator methionyl tRNA anticodon, in order to investigate the effects of base pairing and sequence on the stability of the resulting 43S*mRNA complex. We observed that the formation of three base pairs, rather than their identities, was the key determinant of stability of TC binding, indicating that nothing is inherently special about the sequence AUG for this step. Surprisingly, the rate constant for TC binding to the 40S subunit was strongly codon dependent, whereas the rate constant for TC dissociation from the 43S*mRNA complex was not. The data suggest a model in which, after the initial diffusion-limited encounter of TC with the 40S subunit, the formation of three matching start codon/anticodon base pairs triggers a conformational change that locks the complex into a stable state. This induced-fit mechanism supports the proposal that initiation codon recognition by the 43S complex induces a conformational change from an open state to a closed one that arrests movement along the mRNA.

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

The Molecular Mechanics of Start Site Recognition in Eukaryotic Translation

This talk will describe recent experiments aimed at understanding the molecular mechanisms underlying the recognition of the translation initiation site in an mRNA by the eukaryotic protein synthesis machinery. This is arguably the most important reading of the genetic code during gene expression because if it goes awry a miscoded protein will be produced. Upon recognition of the initiation codon, a signal is sent to the central G protein initiation factor eIF2 to irreversibly hydrolyze its bound GTP and release the methionyl initiator tRNA into the P site of the small ribosomal subunit. This event is the first committed step in translation initiation; after it happens the complex must proceed with initiation at that point on the mRNA or abort the process. Thus the irreversible hydrolysis of GTP by eIF2 must be regulated exquisitely carefully such that it does not happen at the wrong place on the mRNA but happens very rapidly at the right place. We have been exploring the molecular mechanics of the formation of the 43S·mRNA pre-initiation complex, its identification of the start codon in the mRNA, and the triggering of irreversible GTP hydrolysis by eIF2. The work employs a reconstituted yeast translation initiation system, and the roles and mechanisms of each of the key components of the initiation machinery required for these central steps are being elucidated through a thermodynamic and kinetic dissection of the pathway. In collaboration with Alan Hinnebusch's lab at the NIH, we have also been studying a number of mutant versions of initiation factors that produce well characterized phenotypes in vivo, such as reduction in the fidelity of initiation codon recognition. These studies thus synergistically harness the power of yeast genetics and molecular biology to the detailed biophysical and biochemical studies possible in vitro.

GTP-dependent Recognition of the Methionine Moiety on Initiator tRNA by Translation Factor eIF2

Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis. In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNAi) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon. We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2·GTP·Met-tRNAi ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2's affinity for Met-tRNAi in solution. In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNAi that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact. This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway. The toggling on and off of the factor's interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition. In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2. Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2.

Development and characterization of a reconstituted yeast translation initiation system.

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

Coupling of mitochondrial translation with the formation of respiratory complexes in yeast mitochondria.

In contrast to most other eukaryotic organisms, yeast can survive without respiration. This ability has been exploited to investigate nuclear genes required for expression of mitochondrial DNA. Availability of complete Saccharomyces cerevisiae genomic sequence has provided additional help in detailed molecular analysis. Seven of the eight major products encoded by mitochondrial DNA are hydrophobic subunits of respiratory complexes in the inner membrane. Localization of the translation process in the same cellular compartment ensures synthesis of mitochondrially encoded proteins near sites of their assembly into multimeric respiratory complexes. Association of mitochondrial ribosomes with the membrane is mediated by mRNA-specific translational activators, that are involved in the recognition of initiation codon. The newly synthesized mitochondrial proteins are transferred to membrane by a specific export system. This review discusses the role of membrane-localized factors responsible for quality control and turnover of mitochondrially synthesized subunits as well as for assembly of respiratory complexes.