Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.     

A small RNA of Mycoplasma capricolum that resembles eukaryotic U6 small nuclear RNA.

Mycoplasma capricolum, a parasitic prokaryote, contains several small stable RNAs, besides rRNAs and tRNAs. One of them, designated MCS4 RNA (125 nucleotides in length), has been isolated and sequenced. This RNA is abundant in the cell, and is encoded by two genes. Unexpectedly, MCS4 RNA has been found to reveal extensive sequence similarity to eukaryotic U6 snRNAs. This finding suggests that MCS4 and U6 snRNAs are derived from a common ancestral RNA that has existed before the divergence of prokaryotes and eukaryotes.     

Adenosine is inherently favored as the branch-site RNA nucleotide in a structural context that resembles natural RNA splicing

We previously used in vitro selection to identify the 7S11 deoxyribozyme, which catalyzes formation of 2',5'-branched RNA using a branch-site adenosine nucleophile and a 5'-triphosphate electrophile. An unanswered question is whether the use of branch-site adenosine is inherently preferred or a chance event during the particular selection experiment. Here we have found that deoxyribozymes newly selected to use uridine as the branch-site RNA nucleotide in a structural context that resembles natural RNA splicing instead prefer a branch-site adenosine, although adenosine was never available during the selection itself. Our results support a chemical basis for nature's choice of the branch-site nucleotide, which is almost always adenosine in group II introns and the spliceosome.     

6S RNA regulates E. coli RNA polymerase activity

The E. coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive. Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner. UV crosslinking experiments revealed that 6S RNA directly contacts the sigma70 and beta/beta' subunits of RNA polymerase. 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase-specific changes in RNA polymerase. Stable association between sigma70 and core RNA polymerase in extracts is only observed in the presence of 6S RNA. We show 6S RNA represses expression from a sigma70-dependent promoter during stationary phase. Our results suggest that the interaction of 6S RNA with RNA polymerase modulates sigma70-holoenzyme activity.     

Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit

Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.     

HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics

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