The C-terminal domain of the Escherichia coli RNA polymerase subunit plays a role in the CI-dependent activation of the bacteriophage pM promoter (original) (raw)
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Journal of Molecular Biology, 2011
Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP o . The transcription initiation factor, σ 70 , plays critical roles in promoter recognition and RP o formation as well as in early steps of RNA synthesis.
Nucleic Acids Research, 2004
The bacteriophage l CII protein stimulates the activity of three phage promoters, p E , p I and p aQ , upon binding to a site overlapping the ±35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to speci®c residues in the C-terminal domain of the RNA polymerase a subunit (aCTD) to demonstrate that one aCTD binds near position ±41 at p E , whilst the other aCTD binds further upstream. The aCTD bound near position ±41 is oriented such that its 261 determinant is in close proximity to s 70 . The location of aCTD in CII-dependent complexes at the p E promoter is very similar to that found at many activator-independent promoters, and represents an alternative con®guration for aCTD at promoters where activators bind sites overlapping the ±35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of aCTD is involved in stimulation of the p E promoter by CII, and this was con®rmed by in vitro transcription assays. We also show that whereas the K271E substitution in aCTD results in a drastic decrease in CII-dependent activation of p E , the p I and p aQ promoters are less sensitive to this substitution, suggesting that the role of aCTD at the three lysogenic promoters may be different.
Journal of Biological Chemistry
The rrnB P1 promoter of Escherichia coli (starting sequence C"4-A"S-C"2-C"1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Rea. 16,9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3+ " mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1+" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-ternary complexes carry u factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3+ ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
Frontiers in Molecular Biosciences, 2024
Transcription initiation is a multi-step process, in which the RNA polymerase holoenzyme binds to the specific promoter sequences to form a closed complex, which, through intermediate stages, isomerizes into an open complex capable of initiating the productive phase of transcription. The aim of this work was to determine the contribution of the −10 and −35 regions of the promoter, as well as the role of non-specific interactions, in the binding of RNA polymerase and the formation of an active initiation complex capable of transcription. Therefore, fragments of promoter DNA, derived from the strong promoter A1 of the phage T7, containing completely and partially altered elements −35 and −10, and devoid of an upstream region, were constructed using genetic engineering methods. Functional analyses of modified promoter fragments were carried out, checking their ability to form binary complexes with Escherichia coli RNA polymerase (RNAP) and the efficiency of converting binary complexes into triple complexes characteristic of the productive phase of transcription. The obtained results suggest that, in relation to the A1 promoter of the T7 phage, the most important role of the −35 region is carrying the open complex through the next phases of transcription initiation. The weakening of specific impacts within the region −35 is a reason for the defect associated with the transformation of the open complex, formed by a DNA fragment containing the completely altered −35 region, into elongation and the impairment of RNA synthesis. This leads to breaking contacts with the RNA polymerase holoenzyme, and destabilization and disintegration of the complex in the initial phase of productive transcription. This confirms the hypothesis of the so-called stressed intermediate state associated with the stage of transition from the open complex to the elongation complex. The experiments carried out in this work confirm also
Journal of Molecular Biology, 1991
We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of Pa result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which Pa sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the Pa promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the Pa promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the Pz promoter.
The Journal of biological chemistry, 2000
The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state. DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins. Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p(E). The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII. These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations. Examination of the thermodynamic linkages between CII self-association and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4 degree...
Journal of Biological Chemistry
Synthetic 75-base pair promoters bearing base changes and/or base analog substitutions at selected positions were constructed. Using both abortive initiation and run-off transcription assays, the interaction of these altered promoters with Escherichia coli RNA polymerase was studied in order to determine the involvement of DNA functional groups in promoter recognition. Two adjacent thymines in the -35 region were identified whose 5-methyl groups play a crucial role. Additionally, the combined results from several substitution experiments showed that functional groups in the major groove of the strongly conserved T-A base pair at the -7 position are probable sites of direct interaction with RNA polymerase.
Biochemistry, 2003
By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.