Unit: Transcriptional Regulations - CNRS URA 2172

Director: Kolb, Annie

Our laboratory 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 by stressful events such as 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.

Transcription in bacteria is ensured by a multisubunit RNA polymerase composed of the catalytic core enzyme E (subunit composition α2 ββ' w) which associates with one of the multiple σ subunits, responsible for promoter recognition. Each holoenzyme Eσ recognizes a specific set of promoters thus allowing the cell to respond to different environmental stimuli. The E. coli genome encodes seven different σ subunits, which compete for core binding. The association between core and σ is highly regulated under different growth conditions by the availability of core and σ factors and can be modulated by the presence of anti-σ factors.

Promoter selectivity of σS and σ70 RNA polymerases (C. Domecq, S. Lacour, O. Leroy).

In their natural environment bacteria are often exposed to nutrient limitation or stressful conditions. As a consequence they spend most of their time not growing: in this "stationary phase", they are much more resistant to many physical and chemical stresses (pH, temperature, osmolarity, hydrogen peroxide and antibiotics)

Most of the housekeeping genes expressed during exponential growth phase are transcribed by the holoenzyme containing σ 70, the predominant σ factor. As cells reach the end of the exponential phase or are subjected to various environmental stresses, a new σ factor called σ S, or σ 38, comes into play. The stationary phase σ factor σ S is responsible for the transcription of about 100 genes, important for acquisition of the generalized stress resistance status and for survival in the stationary phase. σ S is involved in resistance to osmotic, acid and oxidative stresses, expression of virulence factors and degradation of xenobiotic components.

Among the 6 alternative σ factors of Enterobacteria, σ S is the most similar to σ 70. The similarities are strikingly strong in the promoter binding domains (regions 2.4 and 4.2 of the protein that recognize the -10 and -35 hexamers respectively). Consistently, Eσ S and Eσ 70 bind preferentially almost identical consensus sequences in vitro. However in vivo, specificity is much more stringent. The discrimination mechanism is still not clearly understood: σ S -dependent promoters generally lack a discernible -35 consensus sequence but possess the same optimal -10 hexamer as σ 70-dependent promoters. However unlike Eσ 70, Eσ S might recognize both C and T as first nucleotide in the -10 hexamer and this constitutes a major determinant for selective recognition by Eσ S at several σS -dependent promoters including aidB, a promoter studied in collaboration with P. Landini's laboratory (Dubendorf, Switzerland). Additional promoter features, such as the presence of a TG-motif found at an unusual location and of the C nucleotide immediately upstream of the -10 sequence (C at -13) also represent important factors for σ S -dependent transcription. Using site directed mutagenesis and suppression genetics in collaboration with C. Gutierrez' group in Toulouse, we show that two aminoacids of σ S within region 2.4 (Q152 and E155) participate to the preference for a C at position -13 of its target promoters. Interestingly the homologous residues in σ 70 (Q437 and T440) are also involved in the strong preference for a nucleotide in the -10 region, the first T of the -10 hexamer located at -12 at σ 70-dependent promoters.

Besides promoter sequence elements that determine the intrinsic preference for one or the other holoenzyme, other parameters such as supercoiling or the presence of transcription factors that bind to the promoter region, affect differentially transcription initiation by Eσ s and Eσ 70. They can play an important and sometimes decisive role in determining σ factor selectivity.

Roles of two anti-σ factors Rsd and AsiA :

Anti-σ factors are σ binding regulatory proteins that control the availability and function of various σ factors. Our research focuses on two anti-σ factors which both bind the predominant σ 70 factor: Rsd the stationary phase anti-σ 70 and AsiA encoded by the bacteriophage T4, which show different mechanisms of action.

Competition between σS and σ70 for core RNA polymerase binding (C. Deshayes, C. Domecq, V. Jaumouillé): effect of Rsd

Non growing E coli cells contain more than twice as many σ 70 molecules as σ S molecules. Since core enzyme is limiting and has a higher affinity for σ 70 than for σ S, how σ S can capture sufficient core to assure the expression of σ S-dependent genes in stationary phase is unclear.We are studying different factors that might specifically enhance σ S affinity for core or decrease the affinity of σ 70 for core. Among the latter, the Rsd protein (" Regulator of Sigma D ") expressed upon entry in stationary phase was identified as being able to bind σ70, but not σS. Indeed, in an in vitro competition assay between σS and σ70 in the presence of core, Rsd increases the efficiency of promoter binding by EσS. However, the expression of σ S-dependent promoters in vivo is hardly affected by Rsd, thus suggesting that Rsd is not the determining factor for σ S activity in stationary phase.

AsiA a strong inhibitor of -10/ -35 E. coli promoters has little effect on inhibition of phage T4 early promoters (G. Orsini)

AsiA encoded by the bacteriophage T4 was the first discovered anti-σ factor. Like Rsd, AsiA binds to region 4 of σ 70, but not to σ S. Unlike Rsd which sequesters σ 70 from core, AsiA does not inhibit the formation of the corresponding holoenzyme. However AsiA exerts a much more drastic effect on σ 70 transcription and strongly inhibits σ 70-dependent transcription from bacterial promoters containing "-10/-35 " recognition motives. In contrast AsiA does not prevent promoter complex formation on extended -10 promoters (containing a TG motif at -15/14) most likely because region 4.2 interactions with the -35 element are not required for open complex formation at these promoters. Early T4 promoters are very strong bacterial-like promoters which possess both a highly conserved -35 element and the TG motif together with an UP element which binds the C-terminal domain of the alpha subunits of RNA polymerase. Soon after T4 infection, utilisation of early promoters is abruptly turned off and AsiA has long been suspected to be responsible for this shut-off. In single round transcription we found that most early T4 promoters are markedly resistant to inhibition by AsiA. Either the UP element or the extended -10 site confer resistance to AsiA with a strong preponderance of the UP element in conferring this property to these promoters. Natural modification of the cytosines in bacteriophage T4 DNA (hydroxymethyl glucosyl cytosines) slightly impairs T4 early promoters resistance to AsiA. Our results together with the persistence of a strong shut off of early promoters in the absence of a functional asiA gene which was observed by Pene and Uzan (Institut Jacques Monod-Paris) rule out the idea that AsiA is responsible for the shut off of T4 early promoters.

REGULATION BY THE CRP-cAMP complex (Y. Huo, collaboration with Y-P Wang, Beijing University)

The E. coli cAMP receptor protein (CRP or CAP) was initially identified as an activator of σ 70-dependent transcription at promoters for catabolic operons. However CRP participates in much wider regulatory networks, activating or repressing expression of many genes. It can also modulate the specificity of promoter recognition by different σ factors.

As a class I activator, CRP activates the weak glnAP1 promoter directly from a cis-CRP binding site. In contrast, it can repress the strong glnAP2 promoter, a σ 54 dependent promoter independently of the presence of the upstream CRP binding site. The CRP down-regulation of the activity of σ 54 dependent promoters is studied in collaboration with Y. Huo and Y-P Wang (Beijing University)

Keywords: RNA polymerase, stationary phase, sigma, anti-sigma, AsiA, cyclic AMP Receptor Protein (CRP), MOLECULAR MICROBIOLOGY, BIOCHEMISTRY

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