High-Resolution Structure and Internal Mobility of a Plant 40S Ribosomal Subunit.

Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a limited number of organisms has been resolved to date; it especially concerns plant ribosomes and ribosomal subunits. Here, we report a high-resolution cryo-electron microscopy reconstruction of the small subunit of the Triticum aestivum (common wheat) cytoplasmic ribosome. A detailed atomic model was built that includes the majority of the rRNA and some of the protein modifications. The analysis of the obtained data revealed structural peculiarities of the 40S subunit in the monocot plant ribosome. We applied the 3D Flexible Refinement approach to analyze the internal mobility of the 40S subunit and succeeded in decomposing it into four major motions, describing rotations of the head domain and a shift in the massive rRNA expansion segment. It was shown that these motions are almost uncorrelated and that the 40S subunit is flexible enough to spontaneously adopt any conformation it takes as a part of a translating ribosome or ribosomal complex. Here, we introduce the first high-resolution structure of an isolated plant 40S subunit and the first quantitative analysis of the flexibility of small ribosomal subunits, hoping that it will help in studying various aspects of ribosome functioning.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

RbfA Is Involved in Two Important Stages of 30S Subunit Assembly: Formation of the Central Pseudoknot and Docking of Helix 44 to the Decoding Center.

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.

Unusual dicistronic expression from closely spaced initiation codons in an umbravirus subgenomic RNA.

Translation commencing at closely spaced initiation codons is common in RNA viruses with limited genome space. In the subgenomic RNA (sgRNA) of Pea enation mosaic virus 2, two closely spaced, out-of-frame start codons direct synthesis of movement/stability proteins p26 and p27. Efficient translation from AUG26/AUG27 is dependent on three 3'-proximal cap-independent translation enhancers (3'CITEs), whereas translation of the genomic (gRNA) requires only two. Contrary to strictly scanning-dependent initiation at the gRNA, sequence context of AUG26/AUG27 does not conform with Kozak requirements and insertion of efficient upstream AUGs had pronounced effects for AUG26 but only moderate effects for AUG27. Insertion of a hairpin within an extended 5' UTR did not significantly impact translation from AUG26/AUG27. Furthermore, AUG27 repressed translation from upstream AUG26 and this effect was mitigated when inter-codon spacing was reduced. Addition of a stable hairpin to the very 5' end of the sgRNA severely restricted translation, testifying that this 3'CITE-driven initiation is 5' end-dependent. Similar to gRNA, sgRNA reporter transcripts were nearly exclusively associated with light polysomes and 3'CITE-promoted long-distance interaction connecting the sgRNA ends affected the number of templates translated and not the initiation rate. We propose a non-canonical, 3'CITE-driven mechanism for efficient dicistronic expression from umbravirus sgRNAs.

Functional Cyclization of Eukaryotic mRNAs.

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5' and 3' ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5' untranslated region (UTR) and 3' UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

Concerted action of two 3' cap-independent translation enhancers increases the competitive strength of translated viral genomes.

Several families of plant viruses evolved cap-independent translation enhancers (3'CITE) in the 3' untranslated regions of their genomic (g)RNAs to compete with ongoing cap-dependent translation of cellular mRNAs. Umbravirus Pea enation mosaic virus (PEMV)2 is the only example where three 3'CITEs enhance translation: the eIF4E-binding Panicum mosaic virus-like translational enhancer (PTE) and ribosome-binding 3' T-shaped structure (TSS) have been found in viruses of different genera, while the ribosome-binding kl-TSS that provides a long-distance interaction with the 5' end is unique. We report that the PTE is the key translation promoting element, but inhibits translation in cis and in trans in the absence of the kl-TSS by sequestering initiation factor eIF4G. PEMV2 strongly outcompeted a cellular mRNA mimic for translation, indicating that the combination of kl-TSS and PTE is highly efficient. Transferring the 3'-5' interaction from the kl-TSS to the PTE (to fulfill its functionality as found in other viruses) supported translationin vitro, but gRNA did not accumulate to detectable levels in protoplasts in the absence of the kl-TSS. It was shown that the PTE in conjunction with the kl-TSS did not markedly affect the translation initiation rate but rather increased the number of gRNAs available for translation. A model is proposed to explain how 3'CITE-based regulation of ribosome recruitment enhances virus fitness.

Identification of a Mg2+-sensitive ORF in the 5'-leader of TRPM7 magnesium channel mRNA.

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5'-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes.

Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation.

According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5'-end of mRNA and subsequently migrates along 5'-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5'-UTRs of different lengths and structures in mammalian and plant cell-free systems. For the first time, the relationship between the scanning time and the 5'-UTR length was determined and their linear correlation was experimentally demonstrated. The conclusion is made that the ribosome migration is an unidirectional motion with the rate being virtually independent of a particular mRNA sequence and secondary structure.

Quantitative analysis of ribosome-mRNA complexes at different translation stages.

Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.

Translation of non-capped mRNAs in a eukaryotic cell-free system: acceleration of initiation rate in the course of polysome formation.

Real-time monitoring of the translation of non-capped luciferase mRNA in a wheat germ cell-free system has been performed by continuous in situ measurement of the luminescence increase in the translation mixture. The phenomenon of acceleration of translation has been revealed. It has been shown that the acceleration is accompanied by the loading of translating polysomes with additional ribosomes, and thus is caused mainly by a rise in the initiation rate, rather than the stimulation of elongation or the involvement of additional mRNA molecules in translation. The acceleration requires a sufficient concentration of mRNA and depends on the sequence of the 5' untranslated region (UTR). It can be abolished by the addition of excess cap analog (m(7)GpppGm). As the acceleration does not depend on the preliminary translation of other mRNAs in the same extract, the conclusion has been made that the effect is not due to activation of the ribosome population or other components of the system during translation, but rather it is the consequence of intra-polysomal events. The acceleration observed is discussed in terms of the model of two overlapping initiation pathways in eukaryotic polysomes: translation of non-capped mRNAs starts with eIF4F-independent initiation at 5' UTR, and after the formation of sufficiently loaded polysomes, they rearrange in such a way that a mechanism of re-initiation of terminating ribosomes switches on. The eIF4F-mediated circularization of polysomes may be considered as a possible event that leads to the re-initiation switch and the resultant acceleration effect.

Compilation of tRNA sequences and sequences of tRNA genes.

Maintained at the Universitat Bayreuth, Bayreuth, Germany, the Compilation of tRNA Sequences and Sequences of tRNA Genes is accessible at the URL http://www.tRNA.uni-bayreuth.de with mirror site located at the Institute of Protein Research, Pushchino, Russia (http://alpha.protres.ru/trnadbase). The compilation is a searchable, periodically updated database of currently available tRNA sequences. The present version of the database contains a new Genomic tRNA Compilation including the sequences of tRNA genes from genomic sequences published up to July 2003. It consists of about 5800 tRNA gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya. The former Compilation of tRNA Genes (up to the end of 1998) and the updated Compilation tRNA Sequences (561 entries) are also supported by the new software. The database can be explored by using multiple search criteria and sequence templates. The database provides a service that allows to obtain statistical information on the occurrences of certain bases at given positions of the tRNA sequences. This allows phylogenic studies and search for identity elements in respect to interactions of tRNAs with various enzymes.

Native-like secondary structure of molten globules.

The most common evidence for the existence of secondary structure in a globular protein is the presence of a strongly pronounced far-UV circular dichroism (CD) spectrum. Although CD spectra of native proteins are well described and their quantitative analysis is widely used, similar studies for denatured proteins have still to be done. Far-UV CD spectra of nine proteins in the native and the pH-induced molten globule states were acquired and analyzed. Singular value decomposition showed that the spectra of molten globules could be described as a superposition of at least three independent components (most likely alpha-, beta- and irregular structure). A self-consistent procedure of CD spectra analysis revealed the existence of a clear correlation between the shape of the molten globule spectra and the content of secondary structure elements in the corresponding native proteins, as determined from X-ray data. A mathematical expression of this correlation in terms of the Pierson coefficient amounts to the value of 0.9 for both the alpha-helix and the beta-structure. Thus, the secondary structure of proteins in the molten globule state is close to that in the native state.