RESUMEN
In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estrés Fisiológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fosforilación , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/genética , Biosíntesis de Proteínas , Regulación Fúngica de la Expresión Génica , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Transducción de Señal , Ribosomas/metabolismo , Ribosomas/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genéticaRESUMEN
In plants, pattern-triggered immunity shuts down global translation while allowing the translation of defense mRNAs. Wang et al. (2022) describe a previously unknown mechanism for how elements in the 5' UTR of these mRNAs can recruit the translation machinery to initiate protein synthesis.
Asunto(s)
Biosíntesis de Proteínas , Regiones no Traducidas 5' , ARN Mensajero/genéticaRESUMEN
Stalling during translation triggers ribosome quality control (RQC) to maintain proteostasis. Recently, stalling has also been linked to the activation of integrated stress response (ISR) by Gcn2. How the two processes are coordinated is unclear. Here, we show that activation of RQC by Hel2 suppresses that of Gcn2. We further show that Hel2 and Gcn2 are activated by a similar set of agents that cause ribosome stalling, with maximal activation of Hel2 observed at a lower frequency of stalling. Interestingly, inactivation of one pathway was found to result in the overactivation of the other, suggesting that both are activated by the same signal of ribosome collisions. Notably, the processes do not appear to be in direct competition with each other; ISR prefers a vacant A site, whereas RQC displays no preference. Collectively, our findings provide important details about how multiple pathways that recognize stalled ribosomes coordinate to mount the appropriate response.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Ubiquitina-Proteína Ligasas/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Factores de Tiempo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The recent discovery that collision of ribosomes triggers quality control and stress responses in eukaryotes has shifted the perspective of the field. Collided eukaryotic ribosomes adopt a unique structure, acting as a ubiquitin signaling platform for various response factors. While several of the signals that determine which downstream pathways are activated have been uncovered, we are only beginning to learn how the specificity for the activation of each process is achieved during collisions. This review will summarize those findings and how ribosome-associated factors act as molecular sentinels, linking aberrations in translation to the overall cellular state. Insights into how cells respond to ribosome collision events will provide greater understanding of the role of the ribosome in the maintenance of cellular homeostasis.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/metabolismoRESUMEN
Release factor 3 (RF3) is a GTPase found in a broad range of bacteria where it is thought to play a critical "recycling" role in translation by facilitating the removal of class 1 release factors (RF1 and RF2) from the ribosome following peptide release. More recently, RF3 was shown in vitro to stimulate a retrospective editing reaction on the bacterial ribosome wherein peptides carrying mistakes are prematurely terminated during protein synthesis. Here, we examine the role of RF3 in the bacterial cell and show that the deletion of this gene sensitizes cells to other perturbations that reduce the overall fidelity of protein synthesis. We further document substantial effects on mRNA stability and protein expression using reporter systems, native mRNAs and proteins. We conclude that RF3 plays a primary role in vivo in specifying the fidelity of protein synthesis thus impacting overall protein quantity and quality.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Extensión de la Cadena Peptídica de Translación , Factores de Terminación de Péptidos/metabolismo , Sistema de Lectura Ribosómico , Espectrometría de Masas , Biosíntesis de Proteínas , Estabilidad del ARNRESUMEN
Endogenous and exogenous chemical agents are known to compromise the integrity of RNA and cause ribosome stalling and collisions. Recent studies have shown that collided ribosomes serve as sensors for multiple processes, including ribosome quality control (RQC) and the integrated stress response (ISR). Since RQC and the ISR have distinct downstream consequences, it is of great importance that organisms activate the appropriate process. We previously showed that RQC is robustly activated in response to collisions and suppresses the ISR activation. However, the molecular mechanics behind this apparent competition were not immediately clear. Here we show that Hel2 does not physically compete with factors of the ISR, but instead its ribosomal-protein ubiquitination activity, and downstream resolution of collided ribosomes, is responsible for suppressing the ISR. Introducing a mutation in the RING domain of Hel2-which inhibits its ubiquitination activity and downstream RQC but imparts higher affinity of the factor for collided ribosomes-resulted in increased activation of the ISR upon MMS-induced alkylation stress. Similarly, mutating Hel2's lysine targets in uS10, which is responsible for RQC activation, resulted in increased Gcn4 target induction. Remarkably, the entire process of RQC appears to be limited by the action of Hel2, as the overexpression of this one factor dramatically suppressed the activation of the ISR. Collectively, our data suggest that cells evolved Hel2 to bind collided ribosomes with a relatively high affinity but kept its concentration relatively low, ensuring that it gets exhausted under stress conditions that cannot be resolved by quality control processes.
Asunto(s)
Ribosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estrés Fisiológico , Ubiquitina-Proteína Ligasas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
No-go decay (NGD) is a eukaryotic quality control mechanism that evolved to cope with translational arrests. The process is characterized by an endonucleolytic cleavage near the stall sequence, but the mechanistic details are unclear. Our analysis of cleavage sites indicates that cleavage requires multiple ribosomes on the mRNA. We also show that reporters harboring stall sequences near the initiation codon, which cannot accommodate multiple ribosomes, are not subject to NGD. Consistent with our model, we uncover an inverse correlation between ribosome density per mRNA and cleavage efficiency. Furthermore, promoting global ribosome collision in vivo resulted in ubiquitination of ribosomal proteins, suggesting that collision is sensed by the cell to initiate downstream quality control processes. Collectively, our data suggest that NGD and subsequent quality control are triggered by ribosome collision. This model provides insight into the regulation of quality control processes and the manner by which they reduce off-target effects.
Asunto(s)
Estabilidad del ARN/fisiología , ARN de Hongos/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , ARN de Hongos/genética , Ribosomas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The faithful and rapid translation of genetic information into peptide sequences is an indispensable property of the ribosome. The mechanistic understanding of strategies used by the ribosome to achieve both speed and fidelity during translation results from nearly a half century of biochemical and structural studies. Emerging from these studies is the common theme that the ribosome uses local as well as remote conformational switches to govern induced-fit mechanisms that ensure accuracy in codon recognition during both tRNA selection and translation termination.
Asunto(s)
Escherichia coli/genética , Biosíntesis de Proteínas , Codón/metabolismo , Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Ribosome stalling triggers no-go decay (NGD) and ribosome-associated quality control (RQC) pathways to rapidly degrade the aberrant mRNA and the incomplete nascent peptide, respectively. Two recent studies in yeast and mammalian systems reveal the importance of stalling-induced ribosomal protein ubiquitination by Hel2/ZNF598 for both NGD and RQC The studies also structurally explain how collided ribosomes generate a unique interface not present in monosomes, which can be recognized by Hel2/ZNF598 ubiquitin ligases.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Ubiquitina , Animales , Biosíntesis de Proteínas , Ribosomas , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
In two recent papers, Arenz et al. (2014a) and Bischoff et al. (2014) provide structural insights into drug-induced, peptide-mediated stalling of the ribosome.
Asunto(s)
Eritromicina/química , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/química , Ribosomas/químicaRESUMEN
Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoGâ¢C) and near-cognate (8-oxoGâ¢A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.
Asunto(s)
Emparejamiento Base/genética , Guanosina/análogos & derivados , Conformación de Ácido Nucleico , Ribosomas/genética , Antibacterianos/farmacología , Anticodón/química , Anticodón/genética , Daño del ADN/genética , Escherichia coli/genética , Guanina/química , Guanosina/química , Guanosina/genética , Mutación/efectos de los fármacos , Oxidación-Reducción , ARN Mensajero/genética , ARN de Transferencia , Aminoacil-ARN de Transferencia/efectos de los fármacos , Ribosomas/químicaRESUMEN
During translation, the ribosome plays an active role in ensuring that mRNA is decoded accurately and rapidly. Recently, biochemical studies have also implicated certain accessory factors in maintaining decoding accuracy. However, it is currently unclear whether the mRNA itself plays an active role in the process beyond its ability to base pair with the tRNA. Structural studies revealed that the mRNA kinks at the interface of the P and A sites. A magnesium ion appears to stabilize this structure through electrostatic interactions with the phosphodiester backbone of the mRNA. Here we examined the role of the kink structure on decoding using a well-defined in vitro translation system. Disruption of the kink structure through site-specific phosphorothioate modification resulted in an acute hyperaccurate phenotype. We measured rates of peptidyl transfer for near-cognate tRNAs that were severely diminished and in some instances were almost 100-fold slower than unmodified mRNAs. In contrast to peptidyl transfer, the modifications had little effect on GTP hydrolysis by elongation factor thermal unstable (EF-Tu), suggesting that only the proofreading phase of tRNA selection depends critically on the kink structure. Although the modifications appear to have no effect on typical cognate interactions, peptidyl transfer for a tRNA that uses atypical base pairing is compromised. These observations suggest that the kink structure is important for decoding in the absence of Watson-Crick or G-U wobble base pairing at the third position. Our findings provide evidence for a previously unappreciated role for the mRNA backbone in ensuring uniform decoding of the genetic code.
Asunto(s)
Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/química , Biosíntesis de Proteínas , ARN Mensajero/química , ARN de Transferencia/química , Ribosomas/química , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Electricidad EstáticaRESUMEN
No-go Decay (NGD) is a process that has evolved to deal with stalled ribosomes resulting from structural blocks or aberrant mRNAs. The process is distinguished by an endonucleolytic cleavage prior to degradation of the transcript. While many of the details of the pathway have been described, the identity of the endonuclease remains unknown. Here we identify residues of the small subunit ribosomal protein Rps3 that are important for NGD by affecting the cleavage reaction. Mutation of residues within the ribosomal entry tunnel that contact the incoming mRNA leads to significantly reduced accumulation of cleavage products, independent of the type of stall sequence, and renders cells sensitive to damaging agents thought to trigger NGD. These phenotypes are distinct from those seen in combination with other NGD factors, suggesting a separate role for Rps3 in NGD. Conversely, ribosomal proteins ubiquitination is not affected by rps3 mutations, indicating that upstream ribosome quality control (RQC) events are not dependent on these residues. Together, these results suggest that Rps3 is important for quality control on the ribosome and strongly supports the notion that the ribosome itself plays a central role in the endonucleolytic cleavage reaction during NGD.
Asunto(s)
Estabilidad del ARN , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Genes Fúngicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Extensión de la Cadena Peptídica de Translación , Conformación Proteica , ARN de Hongos/genética , ARN Mensajero/genética , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , UbiquitinaciónRESUMEN
Similar to many other biological molecules, RNA is vulnerable to chemical insults from endogenous and exogenous sources. Noxious agents such as reactive oxygen species or alkylating chemicals have the potential to profoundly affect the chemical properties and hence the function of RNA molecules in the cell. Given the central role of RNA in many fundamental biological processes, including translation and splicing, changes to its chemical composition can have a detrimental impact on cellular fitness, with some evidence suggesting that RNA damage has roles in diseases such as neurodegenerative disorders. We are only just beginning to learn about how cells cope with RNA damage, with recent studies revealing the existence of quality-control processes that are capable of recognizing and degrading or repairing damaged RNA. Here, we begin by reviewing the most abundant types of chemical damage to RNA, including oxidation and alkylation. Focusing on mRNA damage, we then discuss how alterations to this species of RNA affect its function and how cells respond to these challenges to maintain proteostasis. Finally, we briefly discuss how chemical damage to noncoding RNAs such as rRNA, tRNA, small nuclear RNA, and small nucleolar RNA is likely to affect their function.
Asunto(s)
Células/metabolismo , ARN/genética , Animales , Células/citología , Humanos , ARN/metabolismoRESUMEN
Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate's somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.
Asunto(s)
Cromosomas/genética , Proteínas de Unión al ADN/genética , G-Cuádruplex , Proteínas Protozoarias/genética , Tetrahymena thermophila/genética , Proteínas de Unión al ADN/metabolismo , Genoma , Oligonucleótidos/genética , Unión Proteica , Análisis de Secuencia de ADN , Telómero/genéticaRESUMEN
Escherichia coli strains displaying hyperaccurate (restrictive) and ribosomal ambiguity (ram) phenotypes have long been associated with alterations in rpsL and rpsD/rpsE, respectively. Crystallographic evidence shows the ribosomal proteins S12 and S4/S5 (corresponding to these genes) to be located in separate regions of the small ribosomal subunit that are important for domain closure thought to take place during tRNA selection. Mechanistically, the process of tRNA selection is separated into two distinct phases, initial selection and proofreading. Here, we explore the effects of mutations in rpsL and rpsD on these steps. Surprisingly, both restrictive and ram ribosomes primarily affect tRNA selection through alteration of the off rates of the near-cognate tRNA species but during distinct phases of the overall process (proofreading and initial selection, respectively). These observations suggest that closure interfaces (S12/h27/h44 versus S4/S5) in two distinct regions of the small ribosomal subunit function independently to promote high-fidelity tRNA selection.
Asunto(s)
Escherichia coli/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Codón/genética , Activación Enzimática , Proteínas de Escherichia coli , GTP Fosfohidrolasas/metabolismo , Cinética , Mutación/genética , Péptidos/metabolismo , Fenotipo , Estructura Secundaria de Proteína , ARN de Transferencia/genética , Proteína Ribosómica S9 , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m(6)G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m(6)G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m(6)G on the decoding process, using a reconstituted bacterial translation system. m(6)G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyl-tRNAs (aa-tRNAs) by forming m(6)G-uridine codon-anticodon pairs. Surprisingly, the introduction of m(6)G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m(6)A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m(6)G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a Watson-Crick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon-anticodon pairs.
Asunto(s)
Guanosina/análogos & derivados , Aminoacil-ARN de Transferencia/genética , ARN/genética , Ribosomas/química , Ribosomas/metabolismo , Emparejamiento Base , Codón , Guanosina/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Conformación de Ácido Nucleico , ARN/metabolismo , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
The "central dogma" of molecular biology describes how information contained in DNA is transformed into RNA and finally into proteins. In order for proteins to maintain their functionality in both the parent cell and subsequent generations, it is essential that the information encoded in DNA and RNA remains unaltered. DNA and RNA are constantly exposed to damaging agents, which can modify nucleic acids and change the information they encode. While much is known about how cells respond to damaged DNA, the importance of protecting RNA has only become appreciated over the past decade. Modification of the nucleobase through oxidation and alkylation has long been known to affect its base-pairing properties during DNA replication. Similarly, recent studies have begun to highlight some of the unwanted consequences of chemical damage on mRNA decoding during translation. Oxidation and alkylation of mRNA appear to have drastic effects on the speed and fidelity of protein synthesis. As some mRNAs can persist for days in certain tissues, it is not surprising that it has recently emerged that mRNA-surveillance and RNA-repair pathways have evolved to clear or correct damaged mRNA.
Asunto(s)
ARN/metabolismo , Animales , Humanos , Modelos Biológicos , ARN/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismoRESUMEN
In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Terminación de la Cadena Péptídica Traduccional/fisiología , Factores de Terminación de Péptidos/metabolismo , Ribosomas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Factores de Terminación de Péptidos/genética , Ribosomas/genéticaRESUMEN
The ribosome accelerates the rate of peptidyl transfer by >10(6)-fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2'-OH of the peptidyl-tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2'-OH of the peptidyl-tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100-fold slower with the dA76-substituted peptidyl-tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl-tRNA lacks the 2'-OH group. Additionally, the 2'-OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and support a model where interactions between active site residues and the 2'-OH of A76 of the peptidyl-tRNA are pivotal in orienting substrates in this active site for optimal catalysis.