Our lab is interested in transcription initiation, the first step in gene expression at which most of the regulation occurs. In eubacteria this process requires RNA polymerase holoenzyme, composed of the catalytic core enzyme E associated with one of the multiple initiation factor s
, required for promoter recognition.
Promoter selectivity of sS and s70 RNA polymerases (Sandrine Anne, Denise Kotlarz, Anne Lafon and Olivier Leroy).
One of our main interests is to understand how two major s
factors of E. coli , the housekeeping s70 and the stationary phase sS differentially control gene expression. Whereas s70 is constitutively expressed, sS is induced only under stress conditions and at the onset of the stationary phase when it reaches only one third of the s70 level in the cell. The sS regulon includes about 100 genes involved in the protection of the cell against various stresses, the stationary phase survival and the control of virulence. sS is very homologous to s70, especially in the domains involved in the recognition of the promoter. Thus a number of promoters can be recognized in vitro by both sS and s70 RNA polymerase holoenzymes although with different affinities and kinetics. sS -dependent promoters are generally weak promoters which are poorly bound by both RNA polymerases. Most do not contain -35 consensus sequences but a C at position -13. Several mutations that make the promoter sequence closer to the consensus, render the promoter dependent on s70 in vivo
To understand the differential uses of promoters by two s, we have undertaken a comparison of the two holoenzymes alone or when bound to various promoters. Despite a reduced affinity of core for sS, both RNA polymerases show in vitro similar structures and form open complex with similar promoter contacts but in the case of EsS, region 4.2 involved in recognition of the -35 region remains less accessible and more distal from the promoter. At most promoters under study, supercoiling affects differently the two RNA polymerases : E sS is generally less sensitive to a reduction in the level of negative supercoiling than Es70, a finding consistent with the conditions that prevail in the stationary phase. Transcription factors such as CRP-cAMP (the cAMP receptor protein bound to cAMP) , IHF(integration host factor), Lrp (leucine responsive protein) and the silencer HNS differentially affect promoter recognition by both holoenzymes and may even be responsible for the selectivity of the sfactors observed in vivo.
All stationary phase dependent promoters are not dependent on sS. Beatriz Galan (an EMBO student from Pr Garcia's lab CSIC- Madrid) studied the regulation of the s70-dependent hpaB promoter which governs the expression of genes responsible for the catabolism of hydroxyphenyl acetate . Even in the presence of the inducer, this promoter can only be expressed in stationary phase after the complete exhaustion of glucose. This activation requires the CRP-cAMP complex and IHF bound to upstream promoter sequences.
Inhibition of s54-dependent promoters by the CRP-cAMP complex (Pr. Yiping Wang-Peking University)
The CRP-cAMP complex is classically considered as a proximal regulator interacting at short distances with the major form of RNA polymerase, Es
70. Activation also occurs with Es
S (and Es
32) through recruitment of RNA polymerase to the promoter via protein-protein contacts between CRP and the C-terminal part of the alpha subunit , but not with Es
54. To form open complex Es
54 requires an activator of the NtrC family usually bound to distal upstream control sequences and able to hydrolyse XTP.
In contrast CRP-cAMP can exert a negative regulatory function at some s
54-dependent promoters in E. coli, such as glnAp2. glnAp2 expression is activated by nitrogen regulator NtrC-phosphate under the nitrogen limitation and varies strongly when cells grow on different carbohydrates. Primer extension analysis and in vivo permanganate footprinting indicate that glnAp2 downregulation mediated by CRP-cAMP is at the transcriptional level. Site-directed mutagenesis and deletion analysis show that the classical (located at -70.5 of the s70-dependent glnAp1 promoter) and putative CRP binding sites, located at the upstream region of the glnAp2 promoter, are not essential for the CRP inhibitory effect.The cAMP-CRP inhibition remains when the glnAp2 promoter is activated by an alternative activator, NifA.
Inhibition of transcription initiation by the anti- s70 factor AsiA (Gilbert Orsini)
The level and activity of sfactors are also controlled by anti- s factors which prevent core RNA polymerase to associate with s. This is not the case of the AsiA protein, the anti-s factor of bacteriophage T4. In the holoenzyme Es70, AsiA strongly binds to region 4.2 of s70. This is the basis of the dual activity of AsiA. First, it inhibits transcription from bacterial or phage early promoters containing the -35 consensus motif. Secondly, it collaborates with the T4-coded middle transcriptional activator MotA in switching the RNA polymerase holoenzyme Es70 from the early to the middle class of T4 promoters. In order to better understand the AsiA repression mechanism, we have analyzed in the presence of the glutamate anion the properties of the RNA polymerase/AsiA complex relative to the classical -10/-35 bacterial promoter lacUV5. Under these salt conditions widely used in transcription studies, inhibition by AsiA is significantly decreased. The high residual transcriptional activity which is observed is due to the slow transformation of the ternary complex Es70/AsiA/lacUV5 into an active open complex. The affinity of the enzyme for the promoter is 10-fold lower in the ternary complex than in the binary complex Es70/lacUV5. Kinetic analysis of abortive transcription reactions shows that AsiA binding to the holoenzyme results in a 120-fold decrease in the global second order on-rate constant of the reaction of Es70 with lacUV5 with most notably, a 55-fold decrease in the rate constant of the isomerization step leading to the open complex. The active ternary complex still responds to activation by the CRP-cAMP complex, indicating functional protein-protein interactions between CRP and the C-terminal domain of the holoenzyme a subunits. We show that compensatory Es70/promoter upstream contacts involving the C-terminal domain of a subunits in Es70 become essential to allow the productive binding of Es70/AsiA to the lacUV5 promoter.