The functional specificity was compared between two factors, 70 (the major at exponentially growing phase) and 38 (the essential at stationary growth phase), of Escherichia coli RNA polymerase. The core enzyme binding affinity of 38 was less than half the level of 70 as measured by gel filtration column chromatography or by titrating the concentration of required for the maximum transcription in the presence of a fixed amount of core enzyme. In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for E 38 than E 70 . The transcription by E 38 was, however, enhanced with the use of templates with low superhelical density, in good agreement with the decrease in DNA superhelicity in the stationary growth phase. We thus propose that the selective transcription of stationaryspecific genes by E 38 holoenzyme requires either a specific reaction condition(s) or a specific factor(s) such as template DNA with low superhelical density.In Escherichia coli, the total number of RNA polymerase core enzyme is fixed at a level characteristic of the rate of cell growth, which ranges from 1,000 to 3,000 molecules per genome equivalent of DNA (1, 2). On the other hand, the total number of genes on the E. coli genome is estimated to be about 4,000, which is in good agreement with the number estimated from the DNA sequence (up to now, more than 60% has been sequenced). These considerations raise a possibility that competition must take place between promoters for binding a small number of RNA polymerase molecules. Among about 4,000 genes on the E. coli genome, about 1,000 genes are expressed at various levels in exponentially growing cells under laboratory culture conditions, i.e. at 37°C and with aeration (2, 3). The rest of the genes are considered to be expressed under various stress conditions that E. coli meets in nature (4 -7). For instance, a set of stress-response genes is expressed when cells stop growing at stationary phase (6, 7). Transcription of at least some of these stationary phase-specific genes is catalyzed by RNA polymerase holoenzyme containing 38 (the rpoS gene product) (8 -11). In addition, the modification of core enzyme is considered to be involved in stationary-specific transcription regulation (12, 13).Promoters from the stationary-specific genes, however, do not have a single consensus sequence (10,11,14,15). The lack of a consensus could indicate the involvement of a regulatory cascade, in which some genes are directly transcribed by E 38 but others are under the control of these gene products. However, we found that the osmo-regulated genes, osmB and osmY, are transcribed preferentially by E 38 only in the presence of high concentrations of potassium glutamate (or acetate) (16). This finding raises a possibility that each stationary-specific promoter carries a specific sequence that is recognized by E 38 under a specific reaction condition and suggests that the promoter sequences recognized by E 38 differ between gene groups with differe...

Efficient downsizing of peptides: By combination of two orthogonal “conformation‐based” and “sequence‐based” libraries, the cyclic pentapeptide cyclo(‐L‐Nal 1‐Gly 2‐D‐Tyr 3‐L‐Arg 4‐L‐Arg 5‐) (Nal=L‐3‐(2‐naphthyl)alanine; see overlay of the five lowest energy structures), which exhibited strong CXCR4 antagonism (IC50=4 nM) comparable to that of a 14‐residue lead compound, T140, was discovered.

70-dependent transcription in vitro is inhibited at high concentrations of all salts tested, showing maximum activity at 50 mM. In contrast, F -dependent transcription was maximum at 50 mM KCl and then decreased to negligible level at 300 mM; in the cases of potassium acetate and potassium glutamate, maximum transcription was between 200 and 300 mM. DNase I foot printing of the fliC and fliD promoters indicated that F alone is unable to bind DNA, but E F specifically recognizes ؊10 and ؊35 regions of the F -dependent promoters with rather long upstream protection. Alteration of the promoter structure after binding of E F was suggested.

Sequence determinants responsible for promoter recognition by RNA polymerase holoenzyme containing sigma 38, the rpoS gene product, were analyzed. In a previous study [Tanaka et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 3511-3515], Escherichia coli promoters were classified into three groups: promoters recognized only by RNA polymerase holoenzyme containing sigma 70 (E sigma 70); promoters recognized preferentially by that containing sigma 38 (E sigma 38); promoters recognized by both E sigma 70 and E sigma 38. As representatives of each group of promoter, we chose the alaS, fic and lacUV5 promoters. Making use of a restriction enzyme site inserted between the -10 and -35 hexamer sequences, promoters were divided into the upstream (UE) and downstream (DE) elements. These UEs and DEs were combined in all possible combinations and used for in vitro transcription reactions. Promoters containing DE from the fic or lacUV5 promoter were found to be recognized by E sigma 38, while those containing DE from the alaS promoter were not. Moreover, fic DE alone functioned as an efficient promoter for E sigma 38. Thus we conclude that the discrimination signal resides within the DE sequence. To test the activator response of E sigma 38, in vitro transcription reactions were also performed with the gal and lac promoters. For both CRP-responsive P1 promoters, E sigma 38 was found to be activated by the CRP-cAMP complex.