Sandro F. Ataide

The Crystal Structure of the Signal Recognition Particle in Complex with its Receptor.

Guanine triphosphate controls changes in the signal recognition particle that facilitate transfer of the signal sequence to the translocon. Cotranslational targeting of membrane and secretory proteins is mediated by the universally conserved signal recognition particle (SRP). Together with its receptor (SR), SRP mediates the guanine triphosphate (GTP)–dependent delivery of translating ribosomes bearing signal sequences to translocons on the target membrane. Here, we present the crystal structure of the SRP:SR complex at 3.9 angstrom resolution and biochemical data revealing that the activated SRP:SR guanine triphosphatase (GTPase) complex binds the distal end of the SRP hairpin RNA where GTP hydrolysis is stimulated. Combined with previous findings, these results suggest that the SRP:SR GTPase complex initially assembles at the tetraloop end of the SRP RNA and then relocalizes to the opposite end of the RNA. This rearrangement provides a mechanism for coupling GTP hydrolysis to the handover of cargo to the translocon.

Activation of the Pyrrolysine Suppressor tRNA Requires Formation of a Ternary Complex with Class I and Class II Lysyl-tRNA Synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.

The Structural Basis of FtsY Recruitment and GTPase Activation by SRP RNA

The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.

Stationary-phase expression and aminoacylation of a transfer-RNA-like small RNA

Genome‐scale analyses have shown numerous functional duplications in the canonical translational machinery. One of the most striking examples is the occurrence of unrelated class I and class II lysyl‐transfer RNA synthetases (LysRS), which together may aminoacylate non‐canonical tRNAs. We show that, in Bacillus cereus, the two LysRSs together aminoacylate a small RNA of unknown function named tRNAOther, and that the aminoacylated product stably binds translation elongation factor Tu. In vitro reconstitution of a defined lysylation system showed that Lys‐tRNAOther is synthesized in the presence of both LysRSs, but not by either alone. In vivo analyses showed that the class 2 LysRS was present both during and after exponential growth, whereas the class I enzyme and tRNAOther were predominantly produced during the stationary phase. Aminoacylation of tRNAOther was also found to be confined to the stationary phase, which suggests a role for this non‐canonical tRNA in growth‐phase‐specific protein synthesis.

Ribonomic approaches to study the RNA‐binding proteome

Gene expression is controlled through a complex interplay among mRNAs, non‐coding RNAs and RNA‐binding proteins (RBPs), which all assemble along with other RNA‐associated factors in dynamic and functional ribonucleoprotein complexes (RNPs). To date, our understanding of RBPs is largely limited to proteins with known or predicted RNA‐binding domains. However, various methods have been recently developed to capture an RNA of interest and comprehensively identify its associated RBPs. In this review, we discuss the RNA‐affinity purification methods followed by mass spectrometry analysis (AP‐MS); RBP screening within protein libraries and computational methods that can be used to study the RNA‐binding proteome (RBPome).

The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene

Significance In recent years, noncoding RNA transcripts have been found to interact with genes and modulate their ability to be transcribed and made into protein. Here we uncover many of the mechanistic underpinnings involved in how noncoding RNAs control gene transcription. Notably, we find that noncoding RNA control of gene transcription is based on a combination of structural and sequence components of the noncoding RNA and targeted gene. Collectively, the observations presented here suggest that a much more complex and vibrant RNA regulatory world is operative in gene expression and evolution of the genome. RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5′ UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.

A Pseudo-tRNA Modulates Antibiotic Resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNAOther, which does not function in translation. Deletion of tRNAOther led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNAOther. This suggested that tRNAOther might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3′ and 5′-ends of tRNAOther. tRNAOther expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5′ leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNAOther may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.

The CCA anticodon specifies separate functions inside and outside translation in Bacillus cereus

Bacillus cereus 14579 encodes two tRNAs with the CCA anticodon, tRNATrp and tRNAOther. tRNATrp was separately aminoacylated by two enzymes, TrpRS1 and TrpRS2, which share only 34% similarity and display different catalytic capacities and specificities. TrpRS1 was 18-fold more proficient at aminoacylating tRNATrp with Trp, while TrpRS2 more efficiently utilizes the Trp analog 5-hydroxy Trp. tRNAOther was not aminoacylated by either TrpRS but instead by the combined activity of LysRS1 and LysRS2, which recognized sequence elements absent from tRNATrp. Polysomes were found to contain tRNATrp, consistent with its role in translation, but not tRNAOther suggesting a function outside protein synthesis. Regulation of the genes encoding TrpRS1 and TrpRS2 (trpS1 and trpS2) is dependent on riboswitch-mediated recognition of the CCA anticodon, and the role of tRNAOther in this process was investigated. Deletion of tRNAOther led to up to a 50 fold drop in trpS1 expression, which resulted in the loss of differential regulation of the trpS1 and trpS2 genes in stationary phase. These findings reveal that sequence-specific interactions with a tRNA anticodon can be confined to processes outside translation, suggesting a means by which such RNAs may evolve non-coding functions.

RNA and RNA–Protein Complex Crystallography and its Challenges

RNA biology has changed completely in the past decade with the discovery of non-coding RNAs. Unfortunately, obtaining mechanistic information about these RNAs alone or in cellular complexes with proteins has been a major problem. X-ray crystallography of RNA and RNA–protein complexes has suffered from the major problems encountered in preparing and purifying them in large quantity. Here, we review the available techniques and methods in vitro and in vivo used to prepare and purify RNA and RNA–protein complex for crystallographic studies. We also discuss the future directions necessary to explore the vast number of RNA species waiting for their atomic-resolution structure to be determined.

Structural Changes of RNA in Complex with Proteins in the SRP

The structural flexibility of RNA allows it to exist in several shapes and sizes. Thus, RNA is functionally diverse and is known to be involved in processes such as catalysis, ligand binding, and most importantly, protein recognition. RNA can adopt different structures, which can often dictate its functionality. When RNA binds onto protein to form a ribonucleoprotein complex (RNP), multiple interactions and conformational changes occur with the RNA and protein. However, there is the question of whether there is a specific pattern for these changes to occur upon recognition. In particular when RNP complexity increases with the addition of multiple proteins/RNA, it becomes difficult to structurally characterize the overall changes using the current structural determination techniques. Hence, there is a need to use a combination of biochemical, structural and computational modeling to achieve a better understanding of the processes that RNPs are involved. Nevertheless, there are well-characterized systems that are evolutionarily conserved [such as the signal recognition particle (SRP)] that give us important information on the structural changes of RNA and protein upon complex formation.