|Director : Kolb, Annie (firstname.lastname@example.org)|
Our laboratory is interested in two major switches in bacterial gene expression: the transition from exponential to stationary phases where a new sigma factor is synthesised and the early steps of bacteriophage T4 infection where an essential anti-sigma factor encoded by the phage is produced.
RNA polymerase ensures the first step in gene expression and, in all organisms, it is a major target for gene regulation. In eubacteria, the holoenzyme is composed of the core enzyme E (subunit composition a2bb'w) that associates with a s subunit to form the holoenzyme Es able to initiate transcription from promoter sites. Most bacteria contain multiple s factors that play specific roles under various environmental conditions. The concentration of each sigma factor in the cell is tightly controlled by complex mechanisms involving anti-s factors.
Comparison betweens 70AND s S RNA POLYMERASES (S. Anne, A. Kolb, A. Lafon)
The expression of the majority of genes in Escherichia coli is carried out by the housekeeping' s 70 factor but, when bacteria stop growing or are subjected to stress, s 70 is replaced by the alternative stationary phase factor, s S that guides RNA polymerase towards promoters controlling gene products needed for survival and stress resistance. Although the two factors are very similar, they can discriminate between different sets of promoters. The discrimination mechanism is poorly understood, despite the fact that this is an important problem, since most bacteria spend most of their time not growing. Thus our studies focus on the comparison between the housekeeping s 70 and stationary phase s S factors of the common bacterium Escherichia coli.
The two s factors compete for core binding. Even during stationary phase or under stress conditions s S is much less abundant than s 70. Using s tagged at their N- or C-terminus, we showed that the affinity of s S for the core enzyme is ~ 5-fold lower than that of s 70. Hence the question is how in vivo s S can compete with s 70 for core binding.
The two holoenzymes Es S and Es 70 present a similar architecture as shown by Fe-BABE reactivity of single cysteine mutants of s S and s 70. However many s s-dependent promoters do not show any specific - 35 region but most possess a C at position -13, just upstream of the -10 region of the promoter. Thus there is some intrinsic promoter discrimination ability by the two holoenzymes due to promoter sequence and template supercoiling. In addition Es S and Es 70 induce different bending angles in the promoter DNA and respond differentially to intrinsic curvature in the promoter. Moreover transcription factors affect differentially transcription initiation by Es s and Es 70 and can even determine the in vivo s factor selectivity.
Promoter Regulation by the CRP-CAMP complex (B. Galan, D. Kotlarz, Y-P Wang)
The E. coli cAMP receptor protein (CRP or CAP) was initially identified as an activator of s 70-dependent transcription at promoters for catabolic operons. However CRP participates in much wider regulatory networks, activating or repressing expression of many genes, some of them regulated by s S. Depending on the distance between the centre of the CRP binding site, the CRP-cAMP complex can be an activator or a repressor. At the mcc promoter which controls the synthesis of the bacterial protein translation inhibitor microcin C7, a modified heptapeptide induced when cells enter the stationary phase, the CRP-cAMP complex is absolutely required for transcription. In vitro, in the presence of the CRP-cAMP complex, both RNA polymerases Es S and Es 70 transcribe efficiently mcc. However in vivo only Es s is able to alleviate the repression brought about by H-NS a nucleoid protein which acts as a general silencer. (Gonzales-Pastor, San Millan and Moreno, Nature, 1994, 369: 281).
The CRP-cAMP complex is also absolutely required to relieve glucose repression at the hpaG promoter which governs the expression of genes responsible for the catabolism of hydroxyphenyl acetate. Beatriz Galan (an EMBO student from José-Luis Garcia's lab CSIC-Madrid) showed that even in the presence of the inducer, the hpaG 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.
When it acts alone, the CRP-cAMP complex is classically considered as a proximal activator interacting over relatively short distances with Es 70 or Es S RNA polymerases. In contrast Es 54 is regulated by an entirely different mechanism requiring an activator protein that binds distant upstream activator sequences and hydrolyses a nucleoside triphosphate to form open complexes. However Pr Yiping Wang at Beijing University has shown that the CRP-cAMP complex is also able to down-regulate s 54-dependent promoters: for example the glnAp2 promoter is also inhibited. Primer extension and permanganate footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by the heterologous activator NifA, a similar inhibitory effect by CRP-cAMP is observed. The repression effect is eliminated by mutating CRP, or over-expressing the activators or the s 54 factor.
interactions BETWEEN s AND ANTI-s factors (S. Igonet , G. Orsini)
Anti s factors are s binding regulatory proteins that control the availability and function of various s factors. Our research focuses on the anti-s 70 factor AsiA of phage T4. We are interested in the mechanism of action of this factor and in the role of upstream RNA polymerase-promoter contacts in the function of this 10 kDa protein. Several anti-s factors sequester their cognate s and therefore inhibit the formation of the corresponding holoenzyme. AsiA has a different mechanism of action and a dual function:
1- It binds strongly to region 4 of s 70 and inhibits s 70-dependent transcription from bacterial promoters containing " -10 -35 " recognition motives.
2- The s 70-AsiA complex is required in the holoenzyme to activate phage T4 middle transcription that also necessitates the presence of the T4-encoded MotA protein bound to the -30 recognition motif of T4 middle promoters. Together, AsiA and MotA therefore constitute a switching device to change promoter recognition.
We have shown that the binding of AsiA to region 4 of s 70 blocks the functional interaction s 70/ -35 upstream promoter element. Using the glutamate anion to alleviate this blockade, we have analysed in detail the mechanism by which AsiA inhibits transcription from lacUV5, a -10 -35 promoter. Permanganate probing, DNAse I footprinting and laser UV photoreactivity show that the AsiA-containing holoenzyme slowly forms an active open complex at lacUV5. This ternary complex holoenzyme-AsiA-lacUV5 is activated by the CRP-cAMP complex, indicating the importance of upstream polymerase -promoter contacts for this activity. In addition reconstituted RNA polymerase containing a-subunits lacking their carboxy-terminal domain is totally inhibited by AsiA. We are now studying the behaviour of AsiA-containing RNA polymerase relative to several phage T4 early (and very strong) promoters that have an extended upstream curved A/T rich sequence. In this approach, we use AsiA as a probe allowing us to map interactions located in the upstream region of the polymerase promoter complex. The conformational changes brought about by AsiA in s 70 reveal these interactions, which can be identified and analysed.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
Lavenir, Armelle, (email@example.com)
Orsini, Gilbert, (Associate Professor, Université Paris VII, firstname.lastname@example.org)
Igonet, Sébastien, Paris 7 (Student, email@example.com)
Lafon, Anne, Paris 6 (Student, firstname.lastname@example.org)
Wang, Yiping, Beijing University (Professor, email@example.com)
Anne, Sandrine, (Technician, firstname.lastname@example.org)
Kotlarz, Denise, (Engineer, email@example.com)