RNA chaperone activity of large ribosomal subunit proteins from Escherichia coli

The ribosome is a highly dynamic ribonucleoprotein machine. During assembly and during translation the ribosomal RNAs must routinely be prevented from falling into kinetic folding traps. Stable occupation of these trapped states may be prevented by proteins with RNA chaperone activity. Here, ribosomal proteins from the large (50S) ribosome subunit of Escherichia coli were tested for RNA chaperone activity in an in vitro trans splicing assay. Nearly a third of the 34 large ribosomal subunit proteins displayed RNA chaperone activity. We discuss a possible role of this function during ribosome assembly and during translation.     

Messenger RNA movement on the ribosome

Recent X-ray and cryo-EM studies of 70S ribosome complexes containing different types of messenger RNAs (mRNA) and transfer RNA (tRNA) have been reviewed. Changes of the mRNA path on the ribosome at initiation and elongation states have been described. Authors suggested, that the specific region of ribosomal 30S subunit ("platform") is a ribosome binding site of regulatory domains of mRNA which locates on the non-translated 5'-end of the mRNA.     

The translational landscape as regulated by the RNA helicase DDX3

Continuously renewing the proteome, translation is exquisitely controlled by a number of dedicated factors that interact with the ribosome. The RNA helicase DDX3 belonging to the DEAD box family has emerged as one of the critical regulators of translation, the failure of which is frequently observed in a wide range of proliferative, degenerative, and infectious diseases in humans. DDX3 unwinds double-stranded RNA molecules with coupled ATP hydrolysis and thereby remodels complex RNA structures present in various protein-coding and noncoding RNAs. By interacting with specific features on messenger RNAs (mRNAs) and 18S ribosomal RNA (rRNA), DDX3 facilitates translation, while repressing it under certain conditions. We review recent findings underlying these properties of DDX3 in diverse modes of translation, such as cap-dependent and cap-independent translation initiation, usage of upstream open reading frames, and stress-induced ribonucleoprotein granule formation. We further discuss how disease-associated DDX3 variants alter the translation landscape in the cell.     

Protection of ribosomal RNA from kethoxal in polyribosomes: Implication of specific sites in ribosome function

In an attempt to probe the topography of 5 S, 16 S and 23 S RNAs in a functionally engaged ribosome, polysomes were probed using the structure-sensitive, guanine-specifie reagent kethoxal. Reactivities of guanine residues at 38 specific ribosomal RNA sites in polysomes were compared with their corresponding reactivities in vacant 70 S ribosomes. No polysome-specific protection was seen for 5 S RNA. In 16 S RNA, positions 530, 693 or 1079, 966, 1338 and 1517 showed protection in polysomes; all of these sites have highly conserved primary and secondary structures, and include several methylated nucleotides. In 23 S RNA, polysome protection is seen at positions 277, 1071, 1475 or 2112, 2116 and 2751. We attribute polysome-specific protection either to direct contact of transfer RNA and/or messenger RNA with the protected sites or to tRNA and/or mRNA-induced changes in ribosome conformation involving the protected sites.     

Translation initiation: structures, mechanisms and evolution

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.     

线粒体RNA转录后加工和修饰及其与人类线粒体疾病的关联

线粒体是真核细胞内参与能量生成和物质代谢的重要细胞器,拥有自身的基因组DNA.线粒体基因的表达调控对线粒体功能的维持至关重要.根据分子生物学中心法则,遗传信息是从DNA传递给RNA,再从RNA传递给蛋白质.线粒体DNA(mtDNA)编码13个信使RNA(mRNA)、2个核糖体RNA(rRNA)和22个转运RNA(tRN...     

A model for the study of ligand binding to the ribosomal RNA helix h44

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand–RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.     

Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.     

S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA-protein interactions: an NMR and SAXS analysis

The ribosomal protein S1, in Escherichia coli, is necessary for the recognition by the ribosome of the translation initiation codon of most messenger RNAs. It also participates in other functions. In particular, it stimulates the T4 endoribonuclease RegB, which inactivates some of the phage mRNAs, when their translation is no longer required, by cleaving them in the middle of their Shine-Dalgarno sequence. In each function, S1 seems to target very different RNAs, which led to the hypothesis that it possesses different RNA-binding sites. We previously demonstrated that the ability of S1 to activate RegB is carried by a fragment of the protein formed of three consecutive domains (domains D3, D4, and D5). The same fragment plays a central role in all other functions. We analyzed its structural organization and its interactions with three RNAs: two RegB substrates and a translation initiation region. We show that these three RNAs bind the same area of the protein through a set of systematic (common to the three RNAs) and specific (RNA-dependent) interactions. We also show that, in the absence of RNA, the D4 and D5 domains are associated, whereas the D3 and D4 domains are in equilibrium between open (noninteracting) and closed (weakly interacting) forms and that RNA binding induces a structural reorganization of the fragment. All of these results suggest that the ability of S1 to recognize different RNAs results from a high adaptability of both its structure and its binding surface.     

Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites

Studies on the control of eukaryotic translation initiation by a cap-independent recruitment of the 40S ribosomal subunit to internal messenger RNA sequences called internal ribosome entry sites (IRESs) have shown that these sequence elements are present in a growing list of viral and cellular RNAs. Here we discuss their prevalence, mechanisms whereby they may function and their uses in regulating gene expression.     

Structural basis for aminoglycoside inhibition of bacterial ribosome recycling

Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.     

RNA degradation in Saccharomyces cerevisae

All RNA species in yeast cells are subject to turnover. Work over the past 20 years has defined degradation mechanisms for messenger RNAs, transfer RNAs, ribosomal RNAs, and noncoding RNAs. In addition, numerous quality control mechanisms that target aberrant RNAs have been identified. Generally, each decay mechanism contains factors that funnel RNA substrates to abundant exo- and/or endonucleases. Key issues for future work include determining the mechanisms that control the specificity of RNA degradation and how RNA degradation processes interact with translation, RNA transport, and other cellular processes.     

Structural aspects of translation termination on the ribosome

Translation of genetic information encoded in messenger RNAs into polypeptide sequences is carried out by ribosomes in all organisms. When a full protein is synthesized, a stop codon positioned in the ribosomal A site signals termination of translation and protein release. Translation termination depends on class I release factors. Recently, atomic-resolution crystal structures were determined for bacterial 70S ribosome termination complexes bound with release factors RF1 or RF2. In combination with recent biochemical studies, the structures resolve long-standing questions about translation termination. They bring insights into the mechanisms of recognition of all three stop codons, peptidyl-tRNA hydrolysis, and coordination of stop-codon recognition with peptidyl-tRNA hydrolysis. In this review, the structural aspects of these mechanisms are discussed.     

Hepatitis C virus 3′UTR regulates viral translation through direct interactions with the host translation machinery

The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.     

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.