RNA polymerase binding sites in λp lac 5 DNA

Abstract

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of Aplc5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p-, the INT promoter (pI), several sites in the b2 region, the mis promoter, the oop promoter (or po), and pro. Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the CIII-tL region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at PL, at PR9 at a site between R and cos, and at a site at the junction of the y and cIII-tL fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the X genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site). Initiation of mRNA synthesis involves a series of reactions between RNA polymerase holoenzyme (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) and DNA sequences termed "promoters." The steps in this interaction are thought to include (1) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. fined segments by the restriction enzymes HindII and HindIII. RNA polymerase was incubated with the fragments, heparin was added, and the mixture was passed through a nitrocellulose filter, on which the "open complexes" were retained. The DNA fragments associated with RNA polymerase in these complexes were identified by polyacrylamide gel electrophoresis. These complexes were also tested for their initiation potential. Similar types of analyses have been performed previously (2-4).The second technique used is called a "protection experiment" and is identical to one used by Allet and Solem (5) and Allet et al. (6). Some promoters are known to contain HindII sites (6-12) and therefore would not be detected by an RNA polymerase binding experiment. These sites can be discerned by adding RNA polymerase prior to the addition of the HindII and HindIII enzymes and examining the final digest for HindII sites that have not been cleaved. It is assumed that this protection represents specific open complex formation although no competitor has been added and the initiation potential has not been tested.Having identified RNA polymerase...

Correlation of thermodynamic and genetic properties in the Tn10 encoded TET gene control region

“…The denaturation map shown in figure 6 reveals that the bi-directional TET operator-promoter system consisting of two overlapping promoters and possibly two operators [18] is the most unstable part of the 1450-bp sequence. This result agrees well with the notion that promoters tend to occur in AT rich regions of genomic DNA [3,4,201. It is well established that E. coli RNA polymerase unwinds about ten base pairs upstream of the mRNA start nucleotide upon binding to a promoter [51.…”

“…It was noted previously that promotors tend to occur in AT rich regions of the genomic DNA [3,4] which is consistent with the ability of RNA polymerase to unwind about ten base pairs in order to allow the synthesis of a mRNA from the DNA template (5].…”

Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals

“…On the other hand, it has been reported that closed and open complexes are formed in sites that are unproductive for transcription. 33 The resulting scores for the 116 promoters in the training set were rather variable, covering a sevenfold range (1.4 -10.07), being contained within m^3s (see column two in Table 2). The observation described in Figure 3(b) means that the strategy of searching, based on selecting the highest scoring signal, fails in more than 50% of the cases.…”

Genetic analyses of the interactions of the IS 1 -encoded proteins with the left end of IS 1 and its insertion hotspot 1 1Edited by J. Karn

“…The P2 promoter region of pBR322 is in one of the two regions of high A T density in this plasmid as is the P3 promoter, and thus, ÁInsA-B HInsB may have acted on the P2 promoter region. A T-richness is characteristic of RNA polymerase binding sites (Jones et al, 1977;Nakamura & Inoue, 1979), and apparently re¯ects a need for denaturing DNA during transcription initiation. Perhaps IS1 transposition is also facilitated by denaturability of the target site DNA as described by Galas et al (1980).…”