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

Masayori Inouye, Haiping Ke, Ariko Yashio, Kunitoshi Yamanaka, Hirofumi Nariya, Tadashi Shimamoto, Sumiko Inouye

Research output: Contribution to journalArticle

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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 α-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 × 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.

Original languageEnglish (US)
Pages (from-to)50735-50742
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number49
DOIs
StatePublished - Dec 3 2004

Fingerprint

RNA-Directed DNA Polymerase
Thumb
RNA
Escherichia coli
RNA primers
Complementary DNA

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Cite this

Inouye, Masayori ; Ke, Haiping ; Yashio, Ariko ; Yamanaka, Kunitoshi ; Nariya, Hirofumi ; Shimamoto, Tadashi ; Inouye, Sumiko. / Complex formation between a putative 66-residue thumb domain of bacterial reverse transcriptase RT-Ec86 and the primer recognition RNA. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 49. pp. 50735-50742.
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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 α-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 × 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.",
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Complex formation between a putative 66-residue thumb domain of bacterial reverse transcriptase RT-Ec86 and the primer recognition RNA. / Inouye, Masayori; Ke, Haiping; Yashio, Ariko; Yamanaka, Kunitoshi; Nariya, Hirofumi; Shimamoto, Tadashi; Inouye, Sumiko.

In: Journal of Biological Chemistry, Vol. 279, No. 49, 03.12.2004, p. 50735-50742.

Research output: Contribution to journalArticle

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AU - Inouye, Masayori

AU - Ke, Haiping

AU - Yashio, Ariko

AU - Yamanaka, Kunitoshi

AU - Nariya, Hirofumi

AU - Shimamoto, Tadashi

AU - Inouye, Sumiko

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