Complex formation between a putative 66-residue thumb domain of bacterial reverse transcriptase RT-Ec86 and the primer recognition RNA.

The Journal of Biological Chemistry
M InouyeSumiko Inouye

Abstract

Reverse transcriptases (RT) are found in a minor population of Escherichia coli and are responsible for the synthesis of multicopy single-stranded DNA. These RTs specifically recognize RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). Here, we purified the 66-residue, C-terminal fragment of RT-Ec86, RT from E. coli, which is responsible for the synthesis of multicopy single-stranded DNA-Ec86. This fragment, RT-Ec86-(255-320), was found to consist mainly of alpha-helical structures on the basis of its CD spectrum, which is consistent with the prediction of this region as the thumb domain from the structural alignment of RT-Ec86 with human immunodeficiency virus-1 RT. RT-Ec86-(255-320) was able to bind to a 28-base synthetic RNA consisting of the 5'-end single-stranded RNA containing the branching G residue and the recognition stem-loop structure in the RT-Ec86 primer-template RNA with a Kd value of 5 x 10(-8) M. By stepwise shortening of the 5'-end single-stranded region of the RNA, RT-Ec86-(255-320) was found still to be able to form a stable complex with only the stem-loop structure consisting of an 8-bp stem and a 3-b...Continue Reading

References

Jul 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·A Jacobo-MolinaP Clark
Dec 24, 1997·Journal of Bacteriology·J R MaoM Inouye
Jun 11, 1999·The Journal of Biological Chemistry·T A Steitz

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Citations

Oct 11, 2019·Nucleic Acids Research·Anna J SimonIlya J Finkelstein
Jun 25, 2015·Microbiology Spectrum·Steven Zimmerly, Li Wu

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