Conferring RNA polymerase Activity to a DNA polymerase: A single residue in reverse transcriptase controls substrate selection

Abstract

The traditional classification of nucleic acid polymerases as either DNA or RNA polymerases is based, in large part, on their fundamental preference for the incorporation of either deoxyribonucleotides or ribonucleotides during chain elongation. The refined structure determination of Moloney murine leukemia virus reverse transcriptase, a strict DNA polymerase, recently allowed the prediction that a single amino acid residue at the active site might be responsible for the discrimination against the 2OH group of an incoming ribonucleotide. Mutation of this residue resulted in a variant enzyme now capable of acting as an RNA polymerase. In marked contrast to the wild-type enzyme, the K m of the mutant enzyme for ribonucleotides was comparable to that for deoxyribonucleotides. The results are consistent with proposals of a common evolutionary origin for both classes of enzymes and support models of a common mechanism of nucleic acid synthesis underlying catalysis by all such polymerases.A key characteristic of nucleic acid polymerases is their traditional classification as either DNA or RNA polymerases, which is determined by a given enzyme's ability to selectively use either deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs) as substrates for incorporation into a growing chain (1, 2). This classification, however, may not be as fundamental as originally thought (3)(4)(5). Crystallographic studies have demonstrated that DNA and RNA polymerases have remarkable structural similarities (refs. 6-15; reviewed in ref. 16), even though they lack extensive primary sequence homology. Both have a characteristic protein fold forming a nucleic acid binding cleft and a trio of carboxylic acid residues thought to participate directly in catalysis through two bound divalent metal ions. Steady-state analyses further support the notion of a common stepwise polymerization mechanism (17, 18). These observations suggest that it might be possible to convert a DNA polymerase into an RNA polymerase by relatively minor alterations in its structure.Reverse transcriptases (RTs), encoded by all retroviruses, play a defining role in the retroviral life cycle (refs. 19 and 20; for reviews see ref. 21). The enzyme is responsible for the synthesis of a double-stranded linear DNA copy of the RNA genome, which is subsequently inserted into the host genome to form the integrated proviral DNA.The reverse transcription reaction is complex, requiring RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and an associated RNase activity specific for RNA in RNA:DNA hybrid form (22). Although the enzyme can copy either RNA or DNA templates, RT, like all DNA polymerases, can only use deoxyribonucleotides, and not ribonucleotides, as substrates. Studies of the HIV-1 RT have permitted modeling of the position of the incoming nucleotide at the active site (23, 24), with ␣-helices C and E and ␤-sheet strands 6 and 9-11, setting the major topology of the dNTP binding site. A recently determined crystal structure of a catal...

Gln84 of moloney murine leukemia virus reverse transcriptase regulates the incorporation rates of ribonucleotides and deoxyribonucleotides

“…Consistent with our previous results [9], RT-F155V-H could synthesize a stretch of RNA of only seven nucleotides even after 120 min reaction (Fig. 3, lane 6).…”

“…RT-WT-H and RT-F155V-H have been described previously [9,10]. In this report, pTacRT-His-D524N and pTacRT-His-F155V-D524N express RT-WT-H and RT-F155V-H, respectively.…”

“…One unit of RT is defined as the amount of the protein that incorporates 1 nmol of 32 P labeled dTMP in 15 min at 37°C into a form retained on DE81 paper. Kinetic analyses were performed as previously described [9]. The kinetic parameters were determined by double reciprocal plot.…”

“…Substitution of F155 with valine dramatically increased the enzyme's affinity for rNTPs and thereby allowed the enzyme to incorporate rNTPs into the product [9,10]. However, the V max of the RT-F155V mutant enzyme using rUTP as a substrate was little affected, remaining about 100-fold lower than the V max using dTTP as a substrate, and the mutant enzyme could synthesize a stretch of RNA of only seven nucleotides long [9].…”

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

“…Given that the only difference between a dNTP and an rNTP is the absence of a 2Ј OH on the dNTP, it is important that DNA polymerases possess a mechanism to minimize rNTP incorporation into genomic DNA during synthesis. Indeed, most DNA polymerases, including HIV-1 RT, have evolved to discriminate against rNTPs using a bulky residue located in their active site as a steric gate (8,9). HIV-1 RT uses a tyrosine residue at position 115 (Tyr-115) as steric gate, and mutations of this residue have been shown to promote greater rNTP incorporation (10).…”