Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter.

Escherichia coli RNA polymerase (EC 2.7.7.6), bound in a tight complex at an early T7 promoter, protects 41 to 43 base pairs of DNA from digestion by DNase. I. The protected DNA fragment contains both the binding site for RNA polymerase and the mRNA initiation point for the promoter. The sequence of the DNA fragment and the sequence of the mRNA that it codes for are presented here. A seven-base-pair sequence, apparently common to all promoters, is implicated in the formation of a tight binary complex with RNA polymerase.     

Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements.

The 682-base-pair nucleotide sequence between positions 14.45 and 16.15 on the bacteriophage T7 DNA molecule has been determined. We can identify not only the sequence of the primary origin of DNA replication but also the termination of gene 1, all of genes 1.1 and 1.2, the start of gene 1.3, and a number of regulatory sequences. The endpoints of four deletion mutations that extend into this region have been determined. These mutations are inferred to have arisen by recombination between short homologous sequences, three of which ar T7 RNA polymerase promoters. The base changes of four point mutations in gene 1.2 have been identified. The sequence essential for initiation at the primary origin is located between the left endpoints of the two deletions D2 and D303. Sequence analysis of these mutants assigns the primary origin to a 129-base-pair segment between positions 14.73 and 15.05. This intergenic segment is A+T-rich (75%) and contains a single T7 gene 4 protein recognition site; it is preceded by two tandem T7 RNA polymerase promoters. A model for initiation of T7 DNA replication is presented.     

Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7.

Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl-transferase, EC2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before RNA initiation site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Pribnow box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.     

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase.

The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.     

Structure of rat DNA polymerase beta revealed by partial amino acid sequencing and cDNA cloning.

A cDNA library of newborn rat brain poly(A)+ RNA in phage lambda gt11 was screened with a polyclonal antibody against chicken DNA polymerase beta. One positive phage was isolated and purified after testing 2 X 10(7) recombinants. This phage, designated lambda pol beta-10, contained an 1197-base-pair cDNA insert that corresponded to a mRNA with a poly(A) sequence at the 3' terminus and a single, long open-reading frame of 957 bases. The open-reading frame, starting 44 residues from the 5' end of the cDNA, predicted a 36,375-Da protein of 318 amino acids. Comparison of this deduced amino acid sequence with the partial sequence obtained with purified polymerase beta revealed a match of six tryptic peptides, involving a total of 47 amino acid residues. This confirmed the identity of the cDNA. Blot-hybridization analysis of newborn rat brain poly(A)+ RNA revealed a mRNA species of approximately the same size as the cDNA insert; in addition, a second mRNA species approximately equal to 4000 bases long was detected. Computer-derived secondary structure analysis of the enzyme predicted seven regions of alpha-helix distributed throughout and three regions of beta-sheet.     

Interaction of RNA polymerase with forked DNA: evidence for two kinetically significant intermediates on the pathway to the final complex

RNA polymerase forms competitor-resistant complexes with "forked DNA" templates that are double-stranded from the -35 promoter region through the first base pair of the -10 region, with an additional unpaired A at the 3' end of the nontemplate strand. These types of substrates were introduced recently as model templates for the study of DNA-protein interactions occurring in the early stages of the formation of RNA polymerase-promoter open complexes. We have performed kinetic and equilibrium measurements of interactions of wild-type and mutant RNA polymerases bearing substitutions in the sigma(70) initiation factor, with forked DNA of wild-type and mutant sequence. Our observations reveal that formation of a competitor-resistant complex between RNA polymerase and forked DNA, similar to the formation of open complexes at promoters, is a multistep process, and some of the sequentially formed intermediates along the two pathways share common properties. This work establishes, for the forked template, progression through these intermediates in the absence of downstream DNA and validates the use of forked DNA to determine the effects of changes in promoter or RNA polymerase sequence on the process of open complex formation.