188 research outputs found

    Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors

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    Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF45. To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I

    Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

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    The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNAiMet, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNAiMet reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNAiMet, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition

    Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

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    The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNAiMet, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNAiMet reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNAiMet, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition

    Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

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    The HCV internal ribosome entry site (IRES) spans a region of ∼340 nt that encompasses most of the 5′ untranslated region (5′UTR) of the viral mRNA and the first 24–40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5′UTR on IRES activity, naturally occurring variants of the 5′UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation

    Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

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    Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix

    Features of 80S mammalian ribosome and its subunits

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    It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic

    Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade – results of an explorative CGH meta-analysis

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    All available comparative genomic hybridisation (CGH) analyses (n=31, until 12/2003) of human hepatocellular carcinomas (HCCs; n=785) and premalignant dysplastic nodules (DNs; n=30) were compiled and correlated with clinical and histological parameters. The most prominent amplifications of genomic material were present in 1q (57.1%), 8q (46.6%), 6p (22.3%), and 17q (22.2%), while losses were most prevalent in 8p (38%), 16q (35.9%), 4q (34.3%), 17p (32.1%), and 13q (26.2%). Deletions of 4q, 16q, 13q, and 8p positively correlated with hepatitis B virus aetiology, while losses of 8p were more frequently found in hepatitis C virus-negative cases. In poorly differentiated HCCs, 13q and 4q were significantly under-represented. Moreover, gains of 1q were positively correlated with the occurrence of all other high-frequency alterations in HCCs. In DNs, amplifications were most frequently present in 1q and 8q, while deletions occurred in 8p, 17p, 5p, 13q, 14q, and 16q. In conclusion, aetiology and dedifferentiation correlate with specific genomic alterations in human HCCs. Gains of 1q appear to be rather early events that may predispose to further chromosomal abnormalities. Thus, explorative CGH meta-analysis generates novel and testable hypotheses regarding the cause and functional significance of genomic alterations in human HCCs

    Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

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    Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated
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