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  Director : Kolb, Annie (akolb@pasteur.fr)


  abstract

 

Our lab is interested in transcription initiation, the first step of gene expression at which most regulation occurs. Our work is focused on the reprogramming of bacterial expression during two stressful events : the transition from exponential to stationary phase and the early steps of bacteriophage T4 infection. Sigma and anti-sigma factors of RNA polymerase play a key role in these processes.

Bacterial transcription is accomplished by a multisubunit RNA polymerase composed of the catalytic core enzyme (subunit composition a2 b b' w) which associates with one of the multiple s subunits, responsible for promoter recognition. Each holoenzyme Es recognizes a specific set of promoters allowing the cell to respond to different environmental stimuli. The E. coli genome encodes seven different s subunits, which compete for core binding. The association between core and s is highly regulated under different growth conditions by the availability of core and s factors and can be modulated by the presence of anti-s factors.



  report

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Comparison between s70AND sS RNA POLYMERASES (C. Deshayes, A. Kolb, , G. Jan, S. Lacour, E. Marquenet, E. Tate)

Bacteria spend most of their time not growing: in "stationary phase", they are much more resistant to many physical and chemical stresses (changes in pH, temperature, osmolarity, hydrogen peroxide... and antibiotics)

Most of the housekeeping genes expressed during exponential growth phase are transcribed by the holoenzyme containing the principal s factor, s 70. As cells reach the end of the exponential phase or are subjected to various environmental stresses, a new s factor called s S , or s 38 , comes into play. The stationary phase sigma factor s S is responsible for the transcription of many specific genes, important for survival in the stationary phase and for acquisition of the generalized stress resistance status.

The two s factors s S and s 70 are very similar in sequence especially in the DNA binding domains. Nevertheless they can discriminate between different sets of promoters. The discrimination mechanism is poorly understood. s S -dependent promoters are generally weak promoters with no -35 consensus sequences, but a C at position -13. Supercoiling and transcription factors affect differentially transcription initiation by Es s and Es 70 and can even determine the in vivo s factor selectivity.

In vitro most promoters are recognized by both s S and s 70 RNA polymerase holoenzymes although with different affinities and kinetics. Nevertheless in a competition assay containing core, s S and s 70 , the binding discrimination between the two holoenzymes reflects the in vivo situation : the two s factors compete for core binding. s S is much less abundant than s 70 and has a lower affinity for core. Hence the question is how in vivo s S can successfully compete with s 70.

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. The mechanism of action of AsiA is more complex as this protein has 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. Therefore, AsiA and MotA constitute together a switching device designed to change promoter recognition and utilization.

The binding of AsiA to region 4 of s 70 blocks the functional interaction s 70/ -35 upstream promoter element. Using the glutamate anion, this blockade can be partially alleviated and we have analysed in detail the mechanism by which AsiA inhibits transcription from lacUV5, a "-10 -35 " promoter. 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 (up element). Transcription initiated at most of these T4 early promoters is markedly resistant to inhibition in the presence of AsiA. We are currently analyzing the features of the interaction holoenzyme Es 70/AsiA/early promoters which are responsible for this resistance to inhibition, a transcriptional property most likely significant during the development of phage T4.

Promoter Regulation by the CRP-CAMP complex : Collaborations with E. Krin(1), (Institut Pasteur) and Y. Huo and Y-P Wang(2) (Beijing University)

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. In most cases when CRP is the only activator, protein-protein interactions between CRP and RNA polymerase are required to recruit RNA polymerase to the promoter.

1 . ArgG encodes argininosuccinate synthase and is the penultimate gene of the arginine biosynthesis pathway. It is activated by the CRP-cAMP from a CRP binding site located at —167.5 from the transcription start site. ArgG activation does not seem to require protein-protein contacts between CRP and RNA polymerase.

2. An other example of a atypical effect of CRP concerns the down-regulation of s 54 dependent promoters: for instance no matter the presence of an upstream CRP binding site the glnAp2 promoter is inhibited by CRP-cAMP. Primer extension and permanganate footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo.

Keywords: RNA polymerase, stationary phase, sigma, anti-sigma, AsiA, cyclic AMP Receptor Protein (CRP), molecular microbiology, biochemistry



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Lavenir, Armelle, IP, (secretary,alavenir@pasteur.fr) Orsini, Gilbert, Assistant Professor, , Paris VIIorsini@pasteur.fr Tate, Edward, postdoc

Igonet, Sébastien, student Paris VI

Marquenet, Emélie, student Paris VII

Jan, Gaëlle, student, National Agricultural Institute of Paris-Grignon

Huo, Yixin, PhD student, Paris VII and Beijing University, China

Lacour, Stephan, PhD student, cooperation with EAWAG, Zurich, Switzerland

Deshayes, Caroline, student, Paris VII

Legat, Geneviève, Laboratory assistant, IP

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