A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.

About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frameshifting by a mechanism indistinguishable from that of retroviruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.The pol genes of retroviruses are expressed as gag-pol or gag-pro-pol fusion polyproteins (1) formed either by in-frame read-through of termination codons (2, 3) or by ribosomal frameshifting (4-6). Both mechanisms allow for production of multiple proteins from a single, unmodified mRNA.In Rous sarcoma virus (RSV), gag and pol genes overlap, with pol being in the -1 frame with respect to gag (7). In 5% of translations, a -1 frameshifting event allows ribosomes to miss the gag termination codon and continue to translate the pol gene, producing a gag-pol fusion protein (8, 9). A -1 ribosomal frameshifting has also been described in coronaviruses [(+) single-stranded (ss) RNA genomes] (10, 11), phage T7 (12), and in the dnaX gene of Escherichia coli (13)(14)(15). A +1 ribosomal frameshift is seen in the yeast retrotransposon Tyl (16)(17)(18) and in the E. coli release factor 2 (19-21).The signals responsible for -1 ribosomal frameshifting include a "slippery site" heptamer, X XXY YYZ (gag reading frame indicated; X = A, U, or G; Y = A or U; Z = A, U, or C), followed by a stem-loop structure that can be involved in an RNA pseudoknot (4,9,13,20,22,23). A pseudoknot is base pairing of the loop with a sequence 3' of a stem-loop (24, 25). The "simultaneous slippage" model of Jacks et al. (4) proposes that the tRNAs bound at the ribosomal P site to XXY and at the A site to YYZ simultaneously slip back 1 base on the mRNA to pair with XXX and YYY, respectively. Because their nonwobble bases remain properly paired, this can happen at a finite rate (Fig. 1). The stem-loop structure has been demonstrated to be essential for efficient frameshifting in RSV (4), infectious bronchitis virus (23), and the E. coli dnaX gene (13) and is predicted to occur following the slippery site heptamers of a number of other retroviruses (4, 9, 23). RNA secondary structure downstream of the slippery site may slow or stall ribosomes such that they remain in the slippery site longer, thus promoting frameshifting (4).The L-A g...

Summary How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked Dyskeratosis Congenita (X-DC) and Hoyeraal-Hreidarsson syndrome (HH). Here we characterize ribosomes isolated from a yeast strain where Cbf5p, the yeast homologue of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A- and P-sites as well as the cricket paralysis virus IRES (Internal Ribosome Entry Site), which interacts with both the P- and E-sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.

While ribosomes must maintain translational reading frame in order to translate primary genetic information into polypeptides, cis‐acting signals located in mRNAs represent higher order information content that can be used to fine‐tune gene expression. Classes of signals have been identified that direct a fraction of elongating ribosomes to shift reading frame by one base in the 5′ (−1) or 3′ (+1) direction. This is called programmed ribosomal frameshifting (PRF). Although mechanisms of PRF differ, a common feature is induction of ribosome pausing, which alters kinetic partitioning rates between in‐frame and out‐of‐frame codons at specific ‘slippery’ sequences. Many viruses use PRF to ensure synthesis of the correct ratios of virus‐encoded proteins required for proper viral particle assembly and maturation, thus identifying PRF as an attractive target for antiviral therapeutics. In contrast, recent studies indicate that PRF signals may primarily function as mRNA destabilizing elements in cellular mRNAs. These studies suggest that PRF may be used to fine‐tune gene expression through mRNA decay pathways. The possible regulation of PRF by noncoding RNAs is also discussed. WIREs RNA 2012 doi: 10.1002/wrna.1126 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > Computational Analyses of RNA Translation > Translation Regulation

A Brownian machine, a tiny device buffeted by the random motions of molecules in the environment, is capable of exploiting these thermal motions for many of the conformational changes in its work cycle. Such machines are now thought to be ubiquitous, with the ribosome, a molecular machine responsible for protein synthesis, increasingly regarded as prototypical. Here we present a new analytical approach capable of determining the free-energy landscape and the continuous trajectories of molecular machines from a large number of snapshots obtained by cryogenic electron microscopy. We demonstrate this approach in the context of experimental cryogenic electron microscope images of a large ensemble of nontranslating ribosomes purified from yeast cells. The freeenergy landscape is seen to contain a closed path of low energy, along which the ribosome exhibits conformational changes known to be associated with the elongation cycle. Our approach allows model-free quantitative analysis of the degrees of freedom and the energy landscape underlying continuous conformational changes in nanomachines, including those important for biological function.cryo-electron microscopy | elongation cycle | manifold embedding | nanomachines | translation

A new in vivo assay system has been developed to study programmed frameshifting in the yeast Saccharomyces cerevisiae. Frameshift signals are inserted between the Renilla and firefly luciferase reporter genes contained in a yeast expression vector and the two activities are directly measured from cell lysates in one tube. Similar to other bicistronic reporter systems, this one allows the efficient estimation of recoding efficiency by comparison of the normalized activity ratios from each luciferase protein. The assay system has been applied to HIV-1 and L-A directed programmed −1 frameshifting and Ty1 and Ty3 directed +1 frameshifting. The assay system is amenable to high-throughput screening.