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.     

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.     

When needles look like hay: how to find tissue-specific enhancers in model organism genomes

A major prerequisite for the investigation of tissue-specific processes is the identification of cis-regulatory elements. No generally applicable technique is available to distinguish them from any other type of genomic non-coding sequence. Therefore, researchers often have to identify these elements by elaborate in vivo screens, testing individual regions until the right one is found.Here, based on many examples from the literature, we summarize how functional enhancers have been isolated from other elements in the genome and how they have been characterized in transgenic animals. Covering computational and experimental studies, we provide an overview of the global properties of cis-regulatory elements, like their specific interactions with promoters and target gene distances. We describe conserved non-coding elements (CNEs) and their internal structure, nucleotide composition, binding site clustering and overlap, with a special focus on developmental enhancers. Conflicting data and unresolved questions on the nature of these elements are highlighted. Our comprehensive overview of the experimental shortcuts that have been found in the different model organism communities and the new field of high-throughput assays should help during the preparation phase of a screen for enhancers. The review is accompanied by a list of general guidelines for such a project.     

Monoclonal antibodies inhibiting RNA polymerase from Escherichia coli

Monoclonal hybridoma antibodies directed against RNA polymerase from E. coli have been obtained. Only a few have been found to inhibit the enzyme activity. Antibodies produced by two clones, PYN-1 and PYN-2, inhibit RNA polymerase at the stage of RNA chain elongation. The PYN-1 antibodies react with the beta'-subunit of the enzyme. The PYN-2 antibodies react with the beta-subunit and with its 130 kDa amber fragment.     

Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli.

Supercoiled simian virus 40 was transcribed more efficiently than nonsupercoiled DNA. The effect was increased from two- to fivefold by the addition of rifampin with triphosphates. The number and locations of polymerase binding sites with respect to Hin II-III restriction fragments were determined. The total number of binding sites was nine, as determined by UV difference spectroscopy. The locations of these binding sites were on the A, B, D, E, F, and G fragments, as determined by gel electrophoresis. The number of sites was the same for both supercoiled and relaxed or Hin II-III-digested DNA, and the point of saturation of supercoiled DNA by polymerase remained the same with increasing concentrations of rifampin from 0 to 8 microgram/ml.