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Cloning and in vitro characterization of a novel ribosomal frameshifting site in the HIV-1 gag gene

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Rohini Patil (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Ethan Taylor

Abstract: Most retroviruses and various other organisms have a capability of producing multiple protein products from a single RNA species using overlapping genes and taking advantage of ribosomal frameshifting (Jacks T. , 1990; Taylor, 1994). This phenomenon not only helps retroviruses overcome the coding density limitation imposed by their restricted genome size, but also to attain regulatory benefits from the proteins whose genes are present in the overlapping frames, which otherwise is unachievable by normal read through. The classical example is the retroviral pol gene, which in most retroviruses is overlapped by the 3' end of the gag gene, and can only be expressed as a result of -1 ribosomal frameshifting. The pol gene lacks a start codon, and thus can only be expressed via this mechanism. Retroviral -1 ribosomal frameshift sites (RFS) are characterized by two main structural features, an ideal slippery sequence of the pattern "X XXY YYZ", and a pseudoknot present downstream of the slippery site at a distance of up to 10-12 bases from the site. The triplets in the slippery site are the codons represented in the zero frame, and so when a frameshift occurs, the RNA translation machinery shifts by a single base in the 5' direction, where the triplets are then read in the -1 frame as "XXX YYY Z" (Jacks T. , 1988a). Based on this model, several novel frameshift sites have been identified by Taylor et al. in the HIV genome. One such site is a theoretically predicted -1 RFS in the HIV-1 gag gene (EW, 1996). This site (gag-fs), which has not been experimentally validated or studied previously, has become the main focus of this project. A dual reporter assay was employed to study the frameshifting efficiency of the overlapping coding region present in the gag gene (gag-fs) and the constructs required for this assay were successfully made. Based on the results obtained from the assay an estimate of 24% frameshifting efficiency was observed in the wild type when compared to the mutated type (a 100% readthrough control).

Additional Information

Publication
Thesis
Language: English
Date: 2011
Keywords
Retroviruses, Ribosomal frameshifting
Subjects
HIV (Viruses) $x Genetics
Retroviruses $x Genetics
Molecular cloning