Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA

Abstract

A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and $641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution $641A loses RNA polymerase activity but is able to synthesize DNA.Key words: T7 RNA polymerase; Mutagenesis; dNTP utilization; DNA polymerizing activity distribution of hydroxyl-containing amino-acid residues. Specifically, a serine residue is present at position 641 in T7 RNAP (and at corresponding positions in related RNAPs), while in DNA polymerases (DNAP) no such regularity is observed [5]. As $641 is the hydroxyl-bearing amino-acid residue closest to Y639 we have asked whether the hydroxyl groups of these two residues may be involved in the interactions of enzyme with NTP and, specifically, in discrimination between dNTP and rNTP as potential substrates. To test this, we have generated mutant enzymes with phenylalanine in place of tyrosine at position 639, alanine in place of serine in position 641 and a double mutant bearing both of these substitutions. The substrate specificity and other features of the latter two proteins were found to be quite surprising.

On the Evolution of the Single-Subunit RNA Polymerases

“…Sequence motifs found in ssRNAPs and DNAPs do not exist in multisubunit RNAPs (Delarue et al 1990). Moreover, as previously noted, single aminoacid changes are able to lower the ability of the ssRNAP of T7 phage to discriminate between rNTPs and dNTPs (Sousa and Padilla 1995) and can even change the substrate specificity from rNTPs to dNTPs (Kostyuk et al 1995). More recently, Gao et al (1997) have shown that mutation of a single residue in a reverse transcriptase (RT) results in a variant enzyme now capable of acting as an RNAP.…”

“…Another mutation, Y639P, is located at a highly conserved site in these RNAPs (Delarue et al 1990) and disturbs the dNTP/rNTP discrimination of the enzyme (Sousa and Padilla 1995), whereas a S641A mutation directs T7 RNAP to use dNTPs instead of rNTPs (Kostyuk et al 1995). The double mutant Y639F/S641A can use both sets of nucleoside triphosphates (Kostyuk et al 1995).…”

“…The double mutant Y639F/S641A can use both sets of nucleoside triphosphates (Kostyuk et al 1995). Residue Y639 is present in all ssRNAPs, whereas S641 occurs exclusively in the phage enzymes; however, another hydroxy amino acid (T) is found at the adjacent position in all of the available mtRNAP sequences ( Fig.…”

Substrate properties of C′‐methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N‐type NTP conformation

“…The activity of T7 RNAP was assayed according to [7] using plasmids pGEMT [8] or pTZI8R (USB) as substrates. The reaction was carried out for 15 min at 37°C in a mixture containing 40 mM Tris-HCI buffer (pH 7.8), 10 mM MgCb.…”

In Vitro Selection Using Modified or Unnatural Nucleotides

“…In order to improve incorporation, some groups have engineered or evolved polymerases that can incorporate modified nucleotides. For example, a single mutation in T7 RNA polymerase (Y639F) reduces discrimination at the 2′-position and allows more efficient incorporation of deoxyribonucleotides into RNA (Kostyuk et al, 1995;Sousa and Padilla, 1995). Additionally, the Y639F T7 RNA polymerase, which is commercially available in DuraScribe kits (Epicentre, Madison, WI) allows the incorporation of NTPs with fluoro, amino, and thiol groups at their 2′-positions (see Basic Protocol 1).…”