Complex formation between a putative 66-residue thumb domain of bacterial reverse transcriptase RT-Ec86 and the primer recognition RNA.
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-base loop. In this stem-loop structure, the UUU loop was essential for the complex formation. RT-Ec73-(251-316) from another E. coli RT could not bind to the 28-base RNA for RT-Ec86 but could bind to its own stem-loop structure having a 3-base AGU loop. These results support the notion that the highly diverse C-terminal regions of bacterial RTs play an important role in recognizing their own specific primer-template RNA structure for the cDNA priming reaction.
Inouye
Masayori
M
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. inouye@umdnj.edu
Ke
Haiping
H
Yashio
Ariko
A
Yamanaka
Kunitoshi
K
Nariya
Hirofumi
H
Shimamoto
Tadashi
T
Inouye
Sumiko
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D054303:HIV Reverse Transcriptase / Q000737:chemistry; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase