Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation

We have characterized the misincorporation properties of wild-type (wt) T7 RNAP and of 45 T7RNAP point mutants. The wt enzyme selects strongly against incorporation of an incorrect nucleotide. From the measured rates of misincorporation, an average error frequency of 1 in 2 x 10(4) is estimated. RNAs bearing 3'-mismatches are extended more slowly than correctly paired 3'-termini, and mismatches one or two bases away from the RNA 3'-end can also slow extension severely even when the 3'-base is correctly paired. Though it has been reported that T7RNAP has a 3' --> 5' nuclease activity, we were unable to detect any endogenous T7RNAP RNase activity in elongation complexes. Pyrophosphorolysis was detected but does not appear to contribute to proofreading. Therefore, unlike other RNAPs, T7RNAP fidelity appears to depend entirely on discrimination against incorporation of the incorrect nucleotide and not on post-misincorporation proofreading. Alanine substitution of the H784 side chain, which interacts with the 3' RNA.template base pair, increases both misincorporation and mismatch extension, while substitutions at G640, F644, and G645 increase misincorporation, but not mismatch extension. The latter three amino acids are in a part of the RNAP which interacts with the templating base and with the base immediately 5' to the templating base. Mutation of these amino acids not only increases misincorporation, but also eliminates pausing during promoter clearance. The effects of these mutations and the interactions observed in a crystal structure of a transcribing complex indicate that these mutations disrupt interactions which limit misincorporation rates by stabilizing the catalytically incompetent open conformation of the RNAP.     

Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene

A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.     

Isolation and characterization of mutant bacteriophage T7 RNA polymerases.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.     

Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene alpha-tubulin locus. A plasmid construct with P(T7) and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the non-coding strand of an 18SrRNA gene, resulted in tetracycline-inducible expression of the reporter as much as 16- and 150-fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P(T7) augmented the reporter's expression 2-fold more in comparison to P(T7)-less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.     

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.