Book of Abstracts: Albany 2005
Free Dimer of E. coli RNA Polymerase α Subunit is a Potential Transcription Regulator
The E. coli RNA polymerase core enzyme with the subunit composition α2ββ' carries the catalytic function of RNA polymerization. In the sequential pathway of core enzyme assembly, the α subunit forms dimers and then links β and β' polypeptides. To avoid the kinetic limitations in RNA polymerase production, bacterial cells produce α subunits in excess to the RNA polymerase molecules. In the transcription complex, the N-terminal domain of α is involved in subunit-subunit contacts while the C-terminal domain provides contact surfaces with many transcription regulators and promoter UP-elements. Recently it has become clear that free α subunits also have a propensity of interacting with some regulatory factors and DNA having the UP-element-like modules. That means that free α may be involved in transcription regulation affecting expression efficiency of some genes. We tested this possibility by DNA microarrays and found that 2.5 fold over-expression of α alters the spectrum of mRNAs produced in the bacterial cells. The set of genes with altered expression includes fes encoding enterochelin esterase and fepA encoding outer membrane receptor for ferric enterobactin and colicins B and D. These genes are transcribed from divergent and overlapping promoters fes-P and fepA-P1 plus fepA-P2, respectively, and are inhibited by a repressor Fur. Excess α enhanced expression efficiency of fepA and decreased fes transcription.
To examine possible influence of free α on divergent transcription, in vitro studies were performed using a 294 bp PCR fragment containing all three promoters as template. Single round transcription assays confirmed the dependence on α for transcripts produced from promoters fepA-P1 and fes-P. Gel retardation assays testified efficient complex formation of their regulatory regions with RNA polymerase as well as with free α-dimers. The preferred α-subunit binding site was elucidated by DNAse I footprinting. A single α-binding site was identified, which is located in the upstream region of fepA-P1 (positions -73/-48), overlaps with fepA-P2 (positions -45/-20) and lies in the early transcribed region of fes (positions +4/+29). The A/T-rich binding site (AATTTTTC---------GAAATATA) contains two sub-modules separated by several hyper-reactive base pairs to DNAse I. The sequences in the α subunit binding modules form almost ideal inverted repeat, in good agreement with mirror symmetry of two α subunits in the dimer. Complex formation with α may stabilizes RNA polymerase contact with fepA-P1 and interfere with transcription initiation from the promoter fes, thus explaining observed changes in the relative abundance of the corresponding mRNAs. Free α subunits of RNA polymerase may, therefore, provide a possibility to modulate relative transcription efficiency of fepA and fes. To our knowledge this is the first example indicating regulatory potential of free α subunits.
Supported by RFBR grant 03-04-48339 and RFBR-Ministry of Industry and Science (Moscow Region) (grant 04-04-97280).
1Institute of Cell Biophysics RAS