Thermodynamic stability of recoding RNA pseudoknots and ribosomal frameshifting

Publication Year:
2013
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Repository URL:
http://hdl.handle.net/1969.1/ETD-TAMU-2001-Fellows-Thesis-C359
Tags:
biochemistry.; Major biochemistry.
artifact description
Programmed ribosomal frameshifting is a method of regulation of protein expression in which a change in reading frame during translation brings about the formation of an alternative gene product. This process is an important part of the replicative machinery of many RNA viruses, including HIV-1, the causative agent of AIDS. Programmed -1 frameshifting is commonly stimulated via an as yet undetermined mechanism that requires an RNA structural motif in the mRNA coding sequence known as an RNA pseudoknot, found just downstream of the site of -1 slippage. Recently, it was shown that engineering two structural features of the pseudoknots derived from tobacco yellow mosaic virus (a specifically placed loop 2 adenosine), TYMV, and mouse mammary tumor virus (an unpaired adenosine at the stem 1 - stem 2 junction), MMTV could restore efficient frameshifting to a functionally inactive pseudoknot. Thermodynamic analysis presented here shows that a mutant pseudoknot lacking the loop 2 adenosine forms a pseudoknot structure which is stabilized by 1.4 ± 0.5 kcal mol⁻¹. In contrast, two other pseudoknots, in which the junction adenosine residue was substituted with guanosine and the and the other one in which the loop 2 adenosine was replaced with guanosine, both formed moderately less stable structures. All three RNAs display frameshifting efficiencies greatly reduced relative to the wild type RNA. Although these findings suggest that there is no clear correlation between pseudoknot stability and frameshifting efficiency, our data does suggest that a specific interaction between a loop 2 adenosine and helical stem 1 makes a measurable contribution to pseudoknot stability.