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Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications.

Coventry VK, Conn GL - Nucleic Acids Res. (2008)

Bottom Line: In contrast, the Apical Stem unfolds independently and with much higher apparent T(m) of approximately 83 degrees C.Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition.Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNA(I) Central Domain structure essential for PKR inhibitory activity was identified.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, M1 7DN, UK.

ABSTRACT
Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNA(I) were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNA(I) was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent T(m) of approximately 60 degrees C. In contrast, the Apical Stem unfolds independently and with much higher apparent T(m) of approximately 83 degrees C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNA(I) Central Domain structure essential for PKR inhibitory activity was identified.

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Denaturing polyacrylamide gel mobility assay. Wild-type and mutant VA RNAIs were resolved on an 8% polyacrylamide gel containing 50% urea. Size markers (M) correspond to RNAs of 170 and 220 nt.
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Figure 2: Denaturing polyacrylamide gel mobility assay. Wild-type and mutant VA RNAIs were resolved on an 8% polyacrylamide gel containing 50% urea. Size markers (M) correspond to RNAs of 170 and 220 nt.

Mentions: A plasmid for in vitro transcription of Ad2 VA RNAI with a 3′-hepatitis delta virus (HDV) ribozyme was created as described previously (26). Mutant sequences (Table 1 and Figure 1) were generated in this plasmid using the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by automated DNA sequencing. Templates for in vitro transcription reactions with T7 RNA polymerase (T7 RNAP) were prepared by DraI digestion of CsCl gradient purified plasmid DNA. Transcription reactions (0.5–1.0 ml) were performed using 100 μg/ml template DNA under optimal conditions for VA RNAI (27). RNA transcripts were purified by preparative polyacrylamide gel electrophoresis with gels containing 50% urea and 8% acrylamide and eluted from excised bands using a Biotrap device (Schleicher and Schuell). The integrity of the recovered RNA was assessed by urea denaturing PAGE. This process uncovered the electrophoretic mobility differences for one set of mutants (Figure 2).Table 1.


Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications.

Coventry VK, Conn GL - Nucleic Acids Res. (2008)

Denaturing polyacrylamide gel mobility assay. Wild-type and mutant VA RNAIs were resolved on an 8% polyacrylamide gel containing 50% urea. Size markers (M) correspond to RNAs of 170 and 220 nt.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275154&req=5

Figure 2: Denaturing polyacrylamide gel mobility assay. Wild-type and mutant VA RNAIs were resolved on an 8% polyacrylamide gel containing 50% urea. Size markers (M) correspond to RNAs of 170 and 220 nt.
Mentions: A plasmid for in vitro transcription of Ad2 VA RNAI with a 3′-hepatitis delta virus (HDV) ribozyme was created as described previously (26). Mutant sequences (Table 1 and Figure 1) were generated in this plasmid using the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by automated DNA sequencing. Templates for in vitro transcription reactions with T7 RNA polymerase (T7 RNAP) were prepared by DraI digestion of CsCl gradient purified plasmid DNA. Transcription reactions (0.5–1.0 ml) were performed using 100 μg/ml template DNA under optimal conditions for VA RNAI (27). RNA transcripts were purified by preparative polyacrylamide gel electrophoresis with gels containing 50% urea and 8% acrylamide and eluted from excised bands using a Biotrap device (Schleicher and Schuell). The integrity of the recovered RNA was assessed by urea denaturing PAGE. This process uncovered the electrophoretic mobility differences for one set of mutants (Figure 2).Table 1.

Bottom Line: In contrast, the Apical Stem unfolds independently and with much higher apparent T(m) of approximately 83 degrees C.Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition.Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNA(I) Central Domain structure essential for PKR inhibitory activity was identified.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, M1 7DN, UK.

ABSTRACT
Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNA(I) were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNA(I) was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent T(m) of approximately 60 degrees C. In contrast, the Apical Stem unfolds independently and with much higher apparent T(m) of approximately 83 degrees C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNA(I) Central Domain structure essential for PKR inhibitory activity was identified.

Show MeSH
Related in: MedlinePlus