RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

tRNA-like structures tag the 3' ends of genomic RNA molecules for replication: implications for the origin of protein synthesis.

Abstract: Single-stranded RNA viruses often have 3'-terminal tRNA-like structures that serve as substrates for the enzymes of tRNA metabolism, including the tRNA synthases and the CCA-adding enzyme. We propose that such 3'-terminal tRNA-like structures are in fact molecular fossils of the original RNA world, where they tagged genomic RNA molecules for replication and also functioned as primitive telomeres to ensure that 3'-terminal nucleotides were not lost during replication. This picture suggests that the CCA-adding activity was originally an RNA enzyme, that modern DNA telomeres with the repetitive structure CmAn are the direct descendants of the CCA terminus of tRNA, and that the precursor of the modern enzyme RNase P evolved to convert genomic into functional RNA molecules by removing this 3'-terminal tRNA-like tag. Because early RNA replicases would have been catalytic RNA molecules that used the 3'-terminal tRNA-like tag as a template for the initiation of RNA synthesis, these tRNA-like structures could have been specifically aminoacylated with an amino acid by an aberrant activity of the replicase. We show that it is mechanistically reasonable to suppose that this aminoacylation occurred by the same sequence of reactions found in protein synthesis today. The advent of such tRNA synthases would thus have provided a pathway for the evolution of modern protein synthesis.

Laser-mediated, site-specific inactivation of RNA transcripts.

Abstract: The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions In this report, we describe the isolation and in vitro characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation) A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction

Single-molecule analysis of gene expression using two-color RNA labeling in live yeast

Abstract: Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure intrinsic noise in mRNA levels and RNA polymerase II kinetics at a single gene.

Structural basis for RNA replication by the hepatitis C virus polymerase.

Abstract: Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.