Abstract
Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide. Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity. It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity. We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E. coli, and purified the protein to near homogeneity. The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity. These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains.
Full text
PDF












Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Berger S. L., Wallace D. M., Puskas R. S., Eschenfeldt W. H. Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid. Biochemistry. 1983 May 10;22(10):2365–2372. doi: 10.1021/bi00279a010. [DOI] [PubMed] [Google Scholar]
- Carmichael G. G., McMaster G. K. The analysis of nucleic acids in gels using glyoxal and acridine orange. Methods Enzymol. 1980;65(1):380–391. doi: 10.1016/s0076-6879(80)65049-6. [DOI] [PubMed] [Google Scholar]
- Gerard G. F., D'Alessio J. M., Kotewicz M. L., Noon M. C. Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Moloney murine leukemia virus reverse transcriptase. DNA. 1986 Aug;5(4):271–279. doi: 10.1089/dna.1986.5.271. [DOI] [PubMed] [Google Scholar]
- Gerard G. F. Mechanism of action of Moloney murine leukemia virus RNase H III. J Virol. 1981 Feb;37(2):748–754. doi: 10.1128/jvi.37.2.748-754.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gerard G. F. Multiple RNase H activities in mammalian type C retravirus lysates. J Virol. 1978 Apr;26(1):16–28. doi: 10.1128/jvi.26.1.16-28.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gottesman M. E., Adhya S., Das A. Transcription antitermination by bacteriophage lambda N gene product. J Mol Biol. 1980 Jun 15;140(1):57–75. doi: 10.1016/0022-2836(80)90356-3. [DOI] [PubMed] [Google Scholar]
- Grandgenett D. P., Gerard G. F., Green M. A single subunit from avian myeloblastosis virus with both RNA-directed DNA polymerase and ribonuclease H activity. Proc Natl Acad Sci U S A. 1973 Jan;70(1):230–234. doi: 10.1073/pnas.70.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grandgenett D., Quinn T., Hippenmeyer P. J., Oroszlan S. Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains. J Biol Chem. 1985 Jul 15;260(14):8243–8249. [PubMed] [Google Scholar]
- Houts G. E., Miyagi M., Ellis C., Beard D., Beard J. W. Reverse transcriptase from avian myeloblastosis virus. J Virol. 1979 Feb;29(2):517–522. doi: 10.1128/jvi.29.2.517-522.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kanaya S., Crouch R. J. Low levels of RNase H activity in Escherichia coli FB2 rnh result from a single-base change in the structural gene of RNase H. J Bacteriol. 1983 May;154(2):1021–1026. doi: 10.1128/jb.154.2.1021-1026.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kotewicz M. L., D'Alessio J. M., Driftmier K. M., Blodgett K. P., Gerard G. F. Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene. 1985;35(3):249–258. doi: 10.1016/0378-1119(85)90003-4. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lai M. H., Verma I. M. Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity. J Virol. 1978 Feb;25(2):652–663. doi: 10.1128/jvi.25.2.652-663.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lai M. H., Verma I. M., Tronick S. R., Aaronson S. A. Mammalian retrovirus-associated RNase H is virus coded. J Virol. 1978 Sep;27(3):823–825. doi: 10.1128/jvi.27.3.823-825.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levin J. G., Hu S. C., Rein A., Messer L. I., Gerwin B. I. Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure. J Virol. 1984 Aug;51(2):470–478. doi: 10.1128/jvi.51.2.470-478.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
- Moelling K. Characterization of reverse transcriptase and RNase H from friend-murine leukemia virus. Virology. 1974 Nov;62(1):46–59. doi: 10.1016/0042-6822(74)90302-x. [DOI] [PubMed] [Google Scholar]
- Moelling K., Friis R. R. Two avian sarcoma virus mutants with defects in the DNA polymerase-RNase H complex. J Virol. 1979 Nov;32(2):370–378. doi: 10.1128/jvi.32.2.370-378.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Omer C. A., Faras A. J. Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. Cell. 1982 Oct;30(3):797–805. doi: 10.1016/0092-8674(82)90284-7. [DOI] [PubMed] [Google Scholar]
- RICHARDSON C. C., SCHILDKRAUT C. L., APOSHIAN H. V., KORNBERG A. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. XIV. FURTHER PURIFICATION AND PROPERTIES OF DEOXYRIBONUCLEIC ACID POLYMERASE OF ESCHERICHIA COLI. J Biol Chem. 1964 Jan;239:222–232. [PubMed] [Google Scholar]
- Resnick R., Omer C. A., Faras A. J. Involvement of retrovirus reverse transcriptase-associated RNase H in the initiation of strong-stop (+) DNA synthesis and the generation of the long terminal repeat. J Virol. 1984 Sep;51(3):813–821. doi: 10.1128/jvi.51.3.813-821.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roth M. J., Tanese N., Goff S. P. Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J Biol Chem. 1985 Aug 5;260(16):9326–9335. [PubMed] [Google Scholar]