Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase

Wenjing Tong, Chang De Lu, Satish K. Sharma, Suzanne Matsuura, Antero G. So, Walter A. Scott

Research output: Contribution to journalArticle

Abstract

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Dead-end complex was formed between HIV-I RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent K(d) (dTTP <dideoxy-TTP AK α-thio-dTTP <dUTP <3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP <dideoxy-GTP <α- thio-dGTP <<dITP <dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent K(d). By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-I RT are discussed.

Original languageEnglish (US)
Pages (from-to)5749-5757
Number of pages9
JournalBiochemistry®
Volume36
Issue number19
DOIs
StatePublished - May 13 1997
Externally publishedYes

Fingerprint

HIV Reverse Transcriptase
Nucleotides
Zidovudine
RNA-Directed DNA Polymerase
Electrophoresis
Viruses
Dideoxynucleotides
Inosine Triphosphate
Ribonucleotides
Inosine Monophosphate
DNA Primers
Base Pairing
Heparin
Gels
Human immunodeficiency virus 1 reverse transcriptase
thymidine 5'-triphosphate
Substrates
deoxyguanosine triphosphate
triphosphoric acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tong, W., Lu, C. D., Sharma, S. K., Matsuura, S., So, A. G., & Scott, W. A. (1997). Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. Biochemistry®, 36(19), 5749-5757. https://doi.org/10.1021/bi962410z

Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. / Tong, Wenjing; Lu, Chang De; Sharma, Satish K.; Matsuura, Suzanne; So, Antero G.; Scott, Walter A.

In: Biochemistry®, Vol. 36, No. 19, 13.05.1997, p. 5749-5757.

Research output: Contribution to journalArticle

Tong, W, Lu, CD, Sharma, SK, Matsuura, S, So, AG & Scott, WA 1997, 'Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase', Biochemistry®, vol. 36, no. 19, pp. 5749-5757. https://doi.org/10.1021/bi962410z
Tong W, Lu CD, Sharma SK, Matsuura S, So AG, Scott WA. Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. Biochemistry®. 1997 May 13;36(19):5749-5757. https://doi.org/10.1021/bi962410z
Tong, Wenjing ; Lu, Chang De ; Sharma, Satish K. ; Matsuura, Suzanne ; So, Antero G. ; Scott, Walter A. / Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. In: Biochemistry®. 1997 ; Vol. 36, No. 19. pp. 5749-5757.
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