Conformational changes in E. coli RNA polymerase during promoter recognition.

We analysed complexes formed during recognition of the lacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions. In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes. In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.     

Sequence determinants for the recognition of the fork junction DNA containing the -10 region of promoter DNA by E. coli RNA polymerase

It has been recently suggested that E. coli RNA polymerase can specifically recognize a fork junction DNA structure, suggesting a possible role for such interaction in promoter DNA melting [Guo, Y., and Gralla, J. D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11655-11660]. We have determined here quantitatively, using a site-specific binding assay, the effects of base substitutions within the conserved -10 hexamer in the context of a short fork junction DNA on binding to RNA polymerase. Adenine at position -11 and thymine at position -7 were found to be critical for sequence-specific recognition of the DNA. The identities of bases at positions -9 and -8 were found to be not important for the binding whereas replacement of bases at positions -12 and -10 had a mild negative effect on the binding affinity. It was found that for the binding of fork DNA to RNA polymerase, specific sequence recognition was more important than specific recognition of fork junction DNA structure. The pattern of relative importance of bases in the -10 region for binding RNA polymerase was generally consistent with the sequence conservation pattern observed in nature where positions -11 and -7 are the most conserved. Binding experiments with a series of adenine analogues at position -11 revealed that the N1 nitrogen of adenine was a critical determinant for the preference of the adenine at this position, suggesting a mechanism for the nucleation of promoter DNA melting initiation in which RNA polymerase destabilizes duplex DNA by directly competing with the thymine of the A-T base pair for hydrogen bonding to the N1 position of the -11 nontemplate strand adenine.     

How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter

We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.     

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.