The Isolated RNase H Domain of Murine Leukemia Virus Reverse Transcriptase: RETENTION OF ACTIVITY WITH CONCOMITANT LOSS OF SPECIFICITY

Xinyi Zhan,Robert J Crouch

doi:10.1074/jbc.272.35.22023

Abstract

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.

Highlights

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities
E. coli RNase HI, C175-AKR-RNase H, and Chimeric AEA RNase H: Protein Purification—RNases H of E. coli, C175AKR-RNase H, and chimeric AEA were purified from E. coli MIC1066 (rnhA-339::cat recB270(TS)) to ensure that only the overexpressed proteins contribute to the activity observed
We present evidence that a protein comprising the RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) has RNase H activity comparable to the RNase H of AKR-MuLV RT, and it maintains a preference for Mn2ϩ rather than Mg2ϩ

Summary

RETENTION OF ACTIVITY WITH CONCOMITANT LOSS OF SPECIFICITY*

(Received for publication, October 21, 1996, and in revised form, June 25, 1997). Xinyi Zhan and Robert J. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. We show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2؉ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. One construct of E. coli RNase HI was made in which the handle region is missing and in which enzymatic activity is lost due, in part, to a decrease in binding to RNADNA hybrids by more than 3 orders of magnitude (32) In another case, an E. coli RNase HI protein with a different connection of the ␣B and ␣D helices retains some enzymatic activity but only in the presence of Mn2ϩ ions (33).

Isolated RNase H Domain of MuLV Reverse Transcriptase

EXPERIMENTAL PROCEDURES

RESULTS

TABLE I Specific activities using magnesium and manganese

AKR MuLV RT

DISCUSSION