Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter.

Abstract

I have identified those 5 positions of thymines in the lac UV5 promoter that lie close to bound Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Although ultraviolet irradiation of DNA with 5-bromouracil substituted in place of thymine normally cleaves the DNA at the bromouracils, a protein bound to the DNA can perturb these cleavages at those locations at which the protein lies close to the bromine. In the lac promoter most of these contacts lie in three regions. Four contacts lie in the region where transcription initiates; four lie in the "Pribnow box," which is located about 10 base pairs upstream from the initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site. The "Pribnow box" and the "-35 region" are regions whose sequences are partially conserved between promoters and in which most promoter mutations are located; thus, contacts in these two regions probably represent sites of sequence-specific recognition by RNA polymerase. Which features of a promoter direct RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) to initiate RNA synthesis? DNA sequence determination of promoters and promoter mutants provides a partial answer to this question. When the sequences of promoters are compared, two regions of prominent homology have been observed: the "Pribnow box" and the "-35 region" located about 10 and 35 base pairs, respectively, upstream from the start site of transcription (1-4). Furthermore, almost all promoter mutations are located within these regions (5-12).Another approach examines different portions of the promoter for their accessibility to various probes in the presence or absence of RNA polymerase. For example, RNA polymerase bound to any of several promoters protects about 40 base pairs of DNA from endonuclease attack (1,2,13,14). Although polymerase in the resulting complex can initiate correctly and synthesize a transcript about 20 bases long, the 40 base pair fragment does not rebind the polymerase, showing that this fragment lacks some information required for promoter recognition. And, in fact, enzymatic digestion with exonuclease (15) or restriction endonucleases (5,9,(16)(17)(18)(19) suggests that roughly 65 base pairs of DNA are required and that the approximate boundaries of a promoter are located about 45 base pairs upstream and 20 base pairs downstream from the site of initiation. A finer probe is the chemical attack on DNA by dimethyl sulfate (20), which gives a more detailed picture of the interaction because the chemical probe is much smaller than the enzymes that degrade DNA. This probe shows that contacts in one promoter extend from the point of initiation to at least 38 base pairs upstream (21). Because the dimethyl sulfate attacks only the N-7 group of guanine and the N-3 group of adenine on double-stranded DNA, other probes with different specificities are needed to get a complete picture of the crucial promoter elements.I have probed...

Thymine methyls and DNA–protein interactions

“…However, contact with thymine methyls is not actually demonstrated. Hydrogen bonding sites in an A-T basepair at the site could supply the contact point(s) and indirectly quench the cleavage reaction by other means (12).…”

“…Photochemical cleavage of the lac UV5 promoter substituted with shown that at least 10 thymine methyls are covered by RNA polymerase (12,19). Three occur in the -35 site including the two invariant thymines, four occur in the -10 site Including one of the invariant thymines, and three occur in the transcription start site region which is also A-T rich.…”

High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface

“…The operon fusion strain was derived from the deletion strain and is called pop5228 [gehotype: HfrC A(gal-attX-bio) rpoBS00 AmalB107 lacL8UVSp lacZ':: mal'K lamB::lac'Z]. In this fusion strain, gene lamB is expressed from the lacL8UV5p promoter (19,23), which is independent of the cap cyclic AMP control. pop5228 was constructed in two steps.…”

Specific binding and protection of form II SV40 deoxyribonucleic acid by ribonucleic acid polymerase II from wheat germ

“…RNA polymerase II isolated from wheat germ consists of two very large subunits and a pattern of smaller subunits (Jendrisak & Burgess, 1977) which is typical of eucaryotic RNA polymerase II [see Chambon (1975)]. E. coli RNA polymerase also includes among its subunits two very large species, fi and (S', which have been shown to be that part of the core enzyme which may be chemically cross-linked to DNA (Hillel & Wu, 1978; Simpson, 1979). Perhaps the common occurrence of these very large subunits is responsible for the similar binding domains of the procaryotic and eucaryotic binary complexes.…”