Annick Spassky

Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E. Coli RNA polymerase

The nuclease activity of the copper complex of 5-phenyl-1,10-phenanthroline (5-phi-OP-Cu) detects conformational changes in the lac UV-5 promoter caused by E. Coli RNA polymerase. The template strand in melted regions of initiation complexes upstream of the site of nucleotide triphosphate incorporation is very reactive. In open and elongation complexes, downstream scission sites (e.g. +4 and +5 for the open complex) on both strands are observed. The patterns of both downstream cutting sites suggest an atypical double helix at the leading edge of the transcription bubble.

A DNA-binding protein fromE. coliisolation, characterization and its relationship with proteins H1 and B1

A protein of Mr 16,000 (protein 16 K) which tightly binds to DNA has been isolated from E. coli. The amino acid composition and amino-terminal amino acid sequence of this protein have been determined. On the basis of its physico-chemical characteristics the protein 16 K was shown to be identical to the protein H1 [Cukier-Kahn et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3643-3647; Spassky and Buc (1977) Eur. J. Biochem. 81, 79-90] which corresponds itself to the protein B1 [Bakaev (1981) Molekulyarnaya Biologya 15, 1350-1362].

Visualization of the movement of the Escherichia coli RNA polymerase along the Lac UV5 promoter during the initiation of the transcription

Three probes have been used to detect changes in the contacts between Escherichia coli RNA polymerase and lac UV5 promoter during the formation of the open complex and the initiation of the transcription. The results presented here show how contacts between the enzyme and the UV5 promoter are modulated concomitantly with steady-state synthesis of ApApUpU. These results suggest a movement of the enzyme during the initiation of the transcription preceding the irreversible translocation into the elongation complex.

The selection and characterization of two novel mutations in the overlapping promoters of the Escherichia coli galactose operon

Mutations that result in small decreases or increases in expression from the Escherichia coli galactose operon promoter region can be detected by using a plasmid in which the gal promoters were fused to the lac operon. We describe how the level of lac expression was adjusted so that the Lac phenotype of host cells was optimally sensitive to changes in the gal promoter sequence. We have investigated the properties of two new gal promoter mutations both in vivo and in vitro, and have determined their effects on the two overlapping gal promoters, P1 and P2. Although one mutation causes only a small reduction in overall expression in vivo, it completely suppresses transcription initiation at the P1 promoter. However, it also increases expression from the P2 promoter, which compensates for the change at P1. This mutation, a GC to AT transition, falls in a zone just upstream of the P1 Pribnow box, which is essential for P1 activity, whilst improving the homology between the P2 Pribnow box and the consensus sequence. The second mutation causes a small increase in P1 activity. This change, a GC to AT transition at -23, falls in the spacer region between the Pribnow box and the -35 region, a zone containing no known promoter consensus sequences. We suggest that this mutation, which creates a stretch of five AT base pairs, acts by increasing the twist angle of the sequences in the spacer region. We argue that the increase in promoter activity is due to this twist changing the relative orientation of the Pribnow box and -35 regions.

RNA polymerase makes important contacts upstream from base pair −49 at the Escherichia coli galactose operon P1 promoter

A G:C to T:A transversion at bp position -19 in the gal operon promoter region relieves the dependence of galP1 promoter activity on the cAMP-CRP complex. Deletion analysis shows that expression from the promoter is decreased on replacement of the sequence between 49 and 54 bp upstream from the P1 start point. Moreover, protection experiments show that RNA polymerase interacts with this region in open complexes at P1. We propose that this contact is necessary for optimal P1 activity; point mutations in the gal promoter region can alter DNA flexibility and hence the strength of this contact; CRP factor activates P1 transcription by favouring formation of this contact; and the gal repressor blocks P1 activity by binding to this zone.

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein.

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cylic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.

RNA polymerase mutant able to express in vivo and in vitro the lactose operon in the absence of the cAMP-CRP complex

By genetic analysis, we have localized a new mutation, isolated from rho-crp background, responsible for a carbohydrate-positive phenotype. The mutation maps in the rpoB gene coding for the beta-subunit of Escherichia coli RNA polymerase. Using reverse transcriptase analysis of transcripts obtained in vivo and transcription assays in vitro, we have shown that this altered RNA polymerase can efficiently initiate the transcription of the lactose operon in the absence of the cAMP-CRP complex both in vivo and in vitro.