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Functional replacement of two highly conserved tetraloops in the bacterial ribosome.

ABSTRACT: Ribosomes are RNA-protein complexes responsible for protein synthesis. A dominant structural motif in the rRNAs is an RNA helix capped with a four-nucleotide loop, called a tetraloop. The sequence of the tetraloop is invariant at some positions in the rRNAs but is highly variable at other positions. The biological reason for the conservation of the tetraloop sequence at specific positions in the rRNAs is not clear. In the 16S rRNA, the GAAA tetraloop in helix 8 and the UACG tetraloop in helix 14 are highly conserved and located near the binding site for EF-Tu and EF-G. To investigate whether the structural stability of the tetraloop or the precise sequence of the tetraloop is important for function, we separately changed the GAAA tetraloop in helix 8 to a UACG tetraloop and the UACG tetraloop in helix 14 to a GAAA tetraloop. The effects of the tetraloop replacements on protein synthesis were analyzed in vivo and in vitro. Replacement of the tetraloops in helices 8 and 14 did not significantly affect the growth rate of the Escherichia coli (?7rrn) strain. However, the mutant ribosomes showed a slightly reduced rate of protein synthesis in vitro. In addition, we observed a 2-fold increase in the error rate of translation with the mutant ribosomes, which is consistent with an earlier report. Our results suggest that the tetraloops in helices 8 and 14 are highly conserved mainly for their structural stability and the precise sequences of these tetraloops are not critical for protein synthesis.

SUBMITTER: Sahu B

PROVIDER: S-EPMC3786584 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Functional replacement of two highly conserved tetraloops in the bacterial ribosome.

Sahu Bhubanananda B Khade Prashant K PK Joseph Simpson S

Biochemistry 20120912 38

Ribosomes are RNA-protein complexes responsible for protein synthesis. A dominant structural motif in the rRNAs is an RNA helix capped with a four-nucleotide loop, called a tetraloop. The sequence of the tetraloop is invariant at some positions in the rRNAs but is highly variable at other positions. The biological reason for the conservation of the tetraloop sequence at specific positions in the rRNAs is not clear. In the 16S rRNA, the GAAA tetraloop in helix 8 and the UACG tetraloop in helix 14 ...[more]

PMID: 22938718

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Project description:Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition.IMPORTANCE Dengue virus (DENV), an important arthropod-transmitted human pathogen that causes a spectrum of diseases, has spread dramatically worldwide in recent years. Despite extensive efforts, the only commercial vaccine does not provide adequate protection to naive individuals. DENV NS5 polymerase is a promising drug target, as exemplified by the development of successful commercial drugs against hepatitis C virus (HCV) polymerase and HIV-1 reverse transcriptase. High-throughput screening of compound libraries against this enzyme enabled the discovery of inhibitors that induced binding sites in the RNA template channel. Characterizations by biochemical, biophysical, and reverse genetics approaches provide a better understanding of the biological relevance of these allosteric sites and the way forward to design more-potent inhibitors.

Dataset Information

Functional replacement of two highly conserved tetraloops in the bacterial ribosome.

Publications

Functional replacement of two highly conserved tetraloops in the bacterial ribosome.

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