Determination of the promoter strength in the mixed transcription system: promoters of lactose, tryplophan and ribosomal protein L10 operons fromExcherichia Coli

Abstract

In vitro transcription was performed in a single reaction mixture, which contained three species of truncated E. coli DNA template, each carrying one specific promoter, lacP (UV5), trpP or rplJp, and the transcripts of distinct sizes were analyzed by electrophoresis on the same gel. Using this "mixed transcription" system, the order of the promoter strength, i.e., the capacity to form stable open complex, was determined in the single-round transcription under the standard conditions (50 mM NaCl and 37 degrees C) to be lacP greater than trpP greater than rplJp, the latter two promoters being 30--40% and 5--10% the strength of lacP, respectively. After the multiple-round transcription, however, the level of trp transcription was the lowest due to low cyclic-reaction rate but became the highest when another trp fragment containing the natural terminator was used as the template. The order of the transcription level also varied depending on the ionic strength and the reaction temperature and, as a result, lacP was no more the strongest under high salt concentration and at high temperature.

The mediator for stringent control, ppGpp, binds to the β‐subunit of Escherichia coli RNA polymerase

“…Although ribosomal proteins were originally thought to be stringently regulated (Dennis & Nomura 1974), it is now thought that the stringent control effect is direct on rRNA synthesis, but that r-protein production is only affected indirectly via a feedback regulation of translation (Keener & Nomura 1996). Nevertheless, it has been shown on several occasions (Kajitani & Ishihama 1983;Pedersen et al 1984;Nomura et al 1987) that ppGpp inhibits transcription from some of these promoters in vitro and the effect of ppGpp in vitro and in vivo may be different. In spite of rrnBP1 being a strong promoter in vivo, it shows weak transcriptional activity in vitro under normal salt concentrations, and therefore the effect of ppGpp was not detectable under such conditions.…”

“…Similarly, the rplJ promoter fragment prepared by HinfI digestion of the plasmid pJL202 gives a 69 nucleotide transcript (Kajitani & Ishihama 1983). The rpsA fragment was obtained from plasmid pSP261 after Sal I and HinfI digestion and gives a 90 nucleotide transcript (Pedersen et al 1984).…”

Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.

“…The 115-bp BamHIHindIII fragment containing the pstS promoter was obtained from plasmid pOS1 (26), and its ends were filled-in using the Klenow fragment of DNA polymerase I. The 205-bp EcoRI fragment containing the lacUV5 promoter was obtained from plasmid pKB252 (27 The predicted lengths of the run-off transcripts are 45 and 50 nucleotides from the gal P1 and P2 promoters, respectively; 241 nucleotides from the pBR-P4; 98 nucleotides from the ompC promoter; 41 nucleotides from the pstS promoter; and 63 nucleotides from the lacUV5 promoter (see Fig. 1).…”

“…The 115-bp BamHIHindIII fragment containing the pstS promoter was obtained from plasmid pOS1 (26), and its ends were filled-in using the Klenow fragment of DNA polymerase I. The 205-bp EcoRI fragment containing the lacUV5 promoter was obtained from plasmid pKB252 (27). …”

Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif

“…The mutant holoenzymes were purified by Ni-NTA and tested for function in the mixed in vitro transcription system of Ishihama and coworkers using the lacUV5 promoter (Kajitani & Ishihama 1983).…”