Wild-type is the optimal sequence of the HDV ribozyme under cotranscriptional conditions

Durga M. Chadalavada; Andrea L. Cerrone-Szakal; Philip C. Bevilacqua

doi:10.1261/rna.778107

Wild-type is the optimal sequence of the HDV ribozyme under cotranscriptional conditions

Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

Abstract

RNA viruses are responsible for a variety of human diseases, and the pathogenicity of RNA viruses is often attributed to a high rate of mutation. Self-cleavage activity of the wild-type hepatitis delta virus (HDV) ribozyme as measured in standard divalent ion renaturation assays is biphasic and mostly slow and can be improved by multiple rational changes to ribozyme sequence or by addition of chemical denaturants. This is unusual in the sense that wild type is the most catalytically active sequence for the majority of protein enzymes, and RNA viruses are highly mutable. To see whether the ribozyme takes advantage of fast-reacting sequence changes in vivo, we performed alignment of 76 genomic and 269 antigenomic HDV isolates. Paradoxically, the sequence for the ribozyme was found to be essentially invariant in nature. We therefore tested whether three ribozyme sequence changes that improve self-cleavage under standard divalent ion renaturation assays also improve self-cleavage during transcription. Remarkably, wild type was as fast, or faster, than these mutants under cotranscriptional conditions. Slowing the rate of transcription or adding the hepatitis delta antigen protein only further stimulated cotranscriptional self-cleavage activity. Thus, the relative activity of HDV ribozyme mutants depends critically on whether the reaction is assayed under in vivo-like conditions. A model is presented for how wild-type ribozyme sequence and flanking sequence work in concert to promote efficient self-cleavage during transcription. Wild type being the optimal ribozyme sequence under in vivo-like conditions parallels the behavior of most protein enzymes.

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Reprint requests to: Philip C. Bevilacqua, Department of Chemistry, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802, USA; e-mail: pcb{at}chem.psu.edu; fax: (814) 863-8403.
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.778107.
↵1 In this notation, the negative number is how many nucleotides upstream from the cleavage site, which is between −1 and +1, the transcript begins. The positive number is how many nucleotides downstream from the cleavage site the transcript ends, where a transcript with a full-length ribozyme is 85 nt.
↵2 P5 is defined as that portion of the stem–loop downstream of the ribozyme that does not pair with the ribozyme. For functional reasons, its 3′-end boundary is defined by the last nucleotide before the nucleotide that pairs with the 3′ end of the ribozyme. Specifically, P5 runs from C86 to G128, and the attenuator begins at G129 (see Fig. 3). This definition is imperfect because it is possible that mutations that strengthen P5 can still favor unfolding of the ribozyme because of the physical connection between P5 and the attenuator.
↵3 Regions of the attenuator that are complementary to specific secondary structural elements in the ribozyme are denoted with the name of that element preceded with an “a” for attenuator.
- Received August 12, 2007.
- Accepted September 11, 2007.