Mutational analysis of loops 1 and 5 of the hairpin ribozyme

Biochemistry
R ShippyA Hampel

Abstract

A comprehensive analysis of base preferences for all positions in loops 1 and 5 of the hairpin ribozyme-substrate complex was carried out using a cis-ribozyme tethered to substrate by a pentapyrimidine loop. Ribozyme-substrate molecules were mutated to contain each of the three non-native base variations at each of the eight positions within these loops. Catalytic activity was measured for each mutant and compared to the activity of the original native sequence. This was the first time all base positions in these loops have been mutated to all variants and kinetically characterized. Various effects were found, ranging from invariant base positions to those with nearly complete tolerance of any base change. Two positions resulted in cleavage rates below the lower limit of accurate quantification for all non-wild-type base substitutions. These positions are G8 in the ribozyme and Gs6 in the substrate. When A10 was substituted with a pyrimidine, self-cleavage activity fell below the lower limit of detection while the remaining positions showed varying base preferences. The information reported here on loops 1 and 5 combined with previous mutagenesis data on loops 2 and 4 [Siwkowski, A., Shippy, R., and Hampel, A. (1997) Biochemist...Continue Reading

References

Jan 1, 1993·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·D M Long, O C Uhlenbeck

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Citations

Jun 5, 2002·Annual Review of Biochemistry·I-hung Shih, Michael D Been
May 7, 2009·Annual Review of Biophysics·Martha J Fedor
May 24, 2005·Journal of Molecular Biology·Yaroslav I KuzminMartha J Fedor
Mar 25, 2011·Biochemical Society Transactions·David M J Lilley
Jun 18, 2016·Organic & Biomolecular Chemistry·Darko BalkeSabine Müller
Oct 14, 2003·The Journal of Biological Chemistry·Bruno SargueilJohn M Burke
Mar 14, 2007·The Journal of Biological Chemistry·Joseph W CottrellMartha J Fedor
Mar 15, 2000·Journal of Molecular Biology·M J Fedor
Jun 8, 2021·Nucleic Acids Research·Christina E WeinbergZasha Weinberg
Dec 21, 2006·Journal of the American Chemical Society·Jason M Thomas, David M Perrin

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