Random mntagenesis of the gene for bacteriophage T7 RNA polymerase

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.     

Nucleotide sequence of the cloned gene for bacteriophage T7 RNA polymerase

The coding sequence of the gene for bacteriophage T7 RNA polymerase has been cloned into the PstI site of plasmid pBR322. This cloned DNA extends from T7 nucleotide (Dunn & Studier, 1981) 3127 to 5821 or T7 coordinate 7.82 to 14.55. The nucleotide sequence is given, as well as the predicted amino acid sequence of T7 RNA polymerase. This peptide sequence is comprised of 883 amino acid residues with a total molecular weight of 98,092.     

Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

Sequence of bacteriophage T3 DNA from gene 2.5 through gene 9

The nucleotide sequence of bacteriophage T3 DNA, from gene 2.5 through gene 9 has been determined. In addition to regulatory sites, the sequence predicts 19 close-packed genes plus two genes that overlap, in a different reading frame, another gene. The majority of these genes are highly homologous to those in the corresponding region of bacteriophage T7. However, there are some genes that are present in one, but not the other, phage. These apparent deletions are almost exactly gene size and thus the close-packed organization of genes remains the same in T3 as in T7. The varying levels of homology between T3 and T7 DNAs, first noted by Davis and Hyman in their study of DNA heteroduplexes, are also demonstrated here by a comparison of T3 and T7 nucleotide sequences. Many regions of extremely high homology immediately abut sequences that have no apparent homology. These data suggest that bacteriophages T3 and T7 have recombined, both with each other and with other members of a pool of T7-like phages, during their co-evolution.     

Effect of temperature on the transcription by bacteriophage T3-induced RNA polymerase

Bacteriophage T3-induced RNA polymerase is rapidly inactivated at 42 degrees C. Addition of T3 DNA delays this process for 30 s and reduces the rate with which the enzyme activity is lost indicating that a labile binary complex between T3 DNA and polymerase must have been formed. The ternary complex between T3-specific RNA polymerase, T3 DNA, and nascent RNA chains obtained when the enzyme is incubated with T3 DNA, GTP, ATP, and UTP is stable to heat (42 degrees C) and only slowly inactivated by polyvinyl sulfate. The optimal temperature for the formation of polyanionresistant ternary complexes is 30 degrees C while the elongation of T3 RNA chains proceeds fastest at 38 degrees C.     

Modification of RNA polymerase from Escherichia coli by pre-early gene products of bacteriophage T5.

Replication of early region deletion mutations in polyoma in the kidneys of mice was in most cases reduced by 10- to 10,000-fold, as compared with wild-type polyoma, and the mutants failed to produce the persistent infection observed with wild-type polyoma.     

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Sequences of three class II promoters for the bacteriophage T7 RNA polymerase

The sequences of three promoters recognized by the bacteriophage T7 RNA polymerase in the class II region of T7 DNA are reported. They are located at 27.9, 33.3 and 34.7 T7 units. The sequences of these promoters are compared with those of other previously characterized late T7 promoters.