Kelvin Caban

A novel protein domain induces high affinity selenocysteine insertion sequence binding and elongation factor recruitment.

Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec insertion sequence (SECIS) element in their 3′-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNASec, and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site.

The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2

ABSTRACT The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA[Ser]Sec and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.

Selenocysteine Insertion Sequence (SECIS)-binding Protein 2 Alters Conformational Dynamics of Residues Involved in tRNA Accommodation in 80 S Ribosomes

Background: Selenocysteine incorporation requires unique translation factors that interact with the ribosome. Results: SECIS-binding protein 2 alters ribosome conformation at two discrete sites. Conclusion: The selenocysteine incorporation reaction requires ribosome modifications in a region known to be required for tRNA accommodation. Significance: Identifying the ribosomal dynamics required for Sec incorporation will enhance our understanding of the translation elongation reaction. Sec-tRNASec is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNASec is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNASec accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.

Ribosomal Initiation Complex-Driven Changes in the Stability and Dynamics of Initiation Factor 2 Regulate the Fidelity of Translation Initiation

Joining of the large, 50S, ribosomal subunit to the small, 30S, ribosomal subunit initiation complex (IC) during bacterial translation initiation is catalyzed by the initiation factor (IF) IF2. Because the rate of subunit joining is coupled to the IF, transfer RNA (tRNA), and mRNA codon compositions of the 30S IC, the subunit joining reaction functions as a kinetic checkpoint that regulates the fidelity of translation initiation. Recent structural studies suggest that the conformational dynamics of the IF2·tRNA sub-complex forming on the intersubunit surface of the 30S IC may play a significant role in the mechanisms that couple the rate of subunit joining to the IF, tRNA, and codon compositions of the 30S IC. To test this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer signal between IF2 and tRNA that has enabled us to monitor the conformational dynamics of the IF2·tRNA sub-complex across a series of 30S ICs. Our results demonstrate that 30S ICs undergoing rapid subunit joining display a high affinity for IF2 and an IF2·tRNA sub-complex that primarily samples a single conformation. In contrast, 30S ICs that undergo slower subunit joining exhibit a decreased affinity for IF2 and/or a change in the conformational dynamics of the IF2·tRNA sub-complex. These results strongly suggest that 30S IC-driven changes in the stability of IF2 and the conformational dynamics of the IF2·tRNA sub-complex regulate the efficiency and fidelity of subunit joining during translation initiation.

The emerging role of rectified thermal fluctuations in initiator aa-tRNA- and start codon selection during translation initiation

Decades of genetic, biochemical, biophysical, and structural studies suggest that the conformational dynamics of the translation machinery (TM), of which the ribosome is the central component, play a fundamental role in the mechanism and regulation of translation. More recently, single-molecule fluorescence resonance energy transfer (smFRET) studies have provided a unique and powerful approach for directly monitoring the real-time dynamics of the TM. Indeed, smFRET studies of the elongation stage of translation have significantly enriched our understanding of the mechanisms through which stochastic, thermally driven conformational fluctuations of the TM are exploited to drive and regulate the individual steps of translation elongation [1]. Beyond translation elongation, smFRET studies of the conformational dynamics of the initiation stage of translation offer great potential for providing mechanistic information that has thus far remained difficult or impossible to obtain using traditional methods. This is particularly true of the mechanisms through which the accuracy of initiator tRNA- and start codon selection is established during translation initiation. Given that translation initiation is a major checkpoint for regulating the translation of mRNAs, obtaining such mechanistic information holds great promise for our understanding of the translational regulation of gene expression. Here, we provide an overview of the bacterial translation initiation pathway, summarize what is known regarding the biochemical functions of the IFs, and discuss various new and exciting mechanistic insights that have emerged from several recently published smFRET studies of the mechanisms that guide initiator tRNA- and start codon selection during translation initiation. These studies provide a springboard for future investigations of the conformational dynamics of the more complex eukaryotic translation initiation pathway and mechanistic studies of the role of translational regulation of gene expression in human health and disease.

A conformational switch in initiation factor 2 controls the fidelity of translation initiation in bacteria

Initiation factor (IF) 2 controls the fidelity of translation initiation by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNAfMet). Previous studies suggest that rapid 50S subunit joining involves a GTP- and fMet-tRNAfMet-dependent “activation” of IF2, but a lack of data on the structure and conformational dynamics of 30S IC-bound IF2 has precluded a mechanistic understanding of this process. Here, using an IF2-tRNA single-molecule fluorescence resonance energy transfer signal, we directly observe the conformational switch that is associated with IF2 activation within 30S ICs that lack IF3. Based on these results, we propose a model of IF2 activation that reveals how GTP, fMet-tRNAfMet, and specific structural elements of IF2 drive and regulate this conformational switch. Notably, we find that domain III of IF2 plays a pivotal, allosteric, role in IF2 activation, suggesting that this domain can be targeted for the development of novel antibiotics.The GTP-bound form of initiation factor 2 (IF2) promotes translation initiation by accelerating 50S ribosomal subunit joining the 30S ribosomal initiation complex (30S IC). Here the authors use single-molecule FRET and ensemble rapid kinetic methods to uncover the mechanism behind IF2-mediated subunit joining.

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2–6 and 70S5,7–9 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)10 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. We have found that, within ~20–80 ms, rearrangements of the 30S subunit and IF2, uniquely captured in its GDP•Pi-bound state, stabilize fMet-tRNAfMet in its intermediate, ‘70S P/I’, configuration7 and trigger dissociation of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNAfMet into its final, ‘P/P’, configuration within the 70S EC. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.