RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III.

RNase D was recently reported as a new enzymatic activity associated with HIV-1 reverse transcriptase (RT), cleaving RNA at two positions within the double-stranded region of the tRNA primer-viral RNA template complex (Ben-Artzi et al., Proc. Natl. Acad. Sci. USA 89 (1992) 927-931). This would make RNase D a fourth distinct activity of HIV-1 RT, in addition to RNA- and DNA-dependent DNA polymerase and RNase H. Using a specific substrate containing tRNA(Lys,3) hybridized to the primer binding site, we were able to detect the reported RNase D activity in our preparations of recombinant HIV-1 RT. This activity was also present in several active-site mutants of RT, suggesting that it is independent of the RNase H and polymerase functionalities of RT. Furthermore, we found that the cleavage specificity of RNase D is the same as that of RNase III isolated from E.coli. A likely explantation of these results--that the observed RNase D activity is attributable to traces of RNase III contamination--was further strengthened by the finding that the recombinant preparations of HIV-1 RT can specifically cleave a phage T7-derived double-stranded RNA processing signal, which has been used as a model substrate for detection of E.coli RNase III. Moreover, RT purified from an RNase III- strain of E.coli displayed no cleavage of the tRNA primer-RNA template complex.

Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase.

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA.

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.

Structure-function analysis of the mammalian DNA polymerase beta active site: role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer.

The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of KmdTTP, Km(dT)16, and kcat for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure. Mutation of Asp256 to alanine abolished the enzymatic activity of pol beta. Conservative replacement with glutamic acid (D256E) led to a 320-fold reduction of kcat compared to wild type. Replacement of Arg254 with an alanine (R254A) resulted in a 50-fold reduction of kcat compared to wild type. The Km(dT)16 of D256E and R254A increased about 18-fold relative to wild type. Replacement of Arg254 with a lysine resulted in a 15-fold decrease in kcat, and a 5-fold increase in the Km(dT)16. These kinetic observations support a role of Asp256 and Arg254 in the positioning of divalent metal ions and substrates in precise geometrical orientation for efficient catalysis. The mutation of Arg258 to alanine (R258A) resulted in a 10-fold increase in KmdTTP and a 65-fold increase in Km(dT)16 but resulted in no change of kcat. These observations are discussed in the context of the three-dimensional structures of the catalytic domain of pol beta and the ternary complex of pol beta, ddCTP, and DNA.