The relationship between function and DNA sequence in an intercistronic regulatory region in phage λ

Rosenberg, Martin; Court, Donald L.; Shimatake, Hiroyuki; Brady, Catherine; Wulff, Daniel L.

doi:10.1038/272414a0

Cited by 361 publications

(167 citation statements)

References 52 publications

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“…This is characteristic of several transcription termination regions (21) and closely resembles those in the leader segments of the trp operons of E. coli (13), S. typhimurium (13), Shigella dysenteriae (22), and Serratia marcescens (G. F. Miozzari and C. Yanofsky, unpublished results). The occurrence of this termination region in the phe operon accounts for the approximately 140-b leader RNA transcript as the major in vitro transcription product of DNA carrying an intact phe operon promoter and leader region (Fig.…”

Section: Resultsmentioning

confidence: 99%

Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli

Zurawski

Brown

Killingly

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

151

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The isolation of X transducing phages carrying the phenylalanine (phe) operon of Escherichia coli (1) has facilitated the cloning of this operon onto plasmid vectors. We have mapped the phe operon by cleavage with restriction endonucleases and by genetic analysis. In this paper we report that there is a transcribed leader region of about 170 base pairs (bp) immediately preceding pheA, the sole structural gene of the phe operon. The function of this region in the regulation of phe operon expression is discussed. MATERIALS AND METHODSConstruction and Preparation of Plasmids. The methods used for ligation, transformation, and preparation of plasmid DNAs were essentially those described in Selker et al. (2).Restriction Mapping. The methods used for restriction endonuclease digestion and analysis of DNA fragments by agarose and polyacrylamide gel electrophoresis have been described (3). A detailed restriction analysis of the phe operon will appear elsewhere.In Vitro Transcription. Plasmids and restriction fragments were transcribed and the 32P-labeled RNA products were analyzed by electrophoresis on 10% polyacrylamide/Tris borate/EDTA gels containing 7M urea as described (4).

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Section: Resultsmentioning

confidence: 99%

Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli

Zurawski

Brown

Killingly

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

151

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“…To test this hypothesis we examined the level of readthrough at rightward terminators in an N ¹ system. The t R1 site is a weak -dependent terminator (Court et al, 1980) and a significant proportion (15-50%) of transcribing RNA polymerase molecules are able to proceed beyond this region (Dambly et al, 1976;Rosenberg et al, 1978;Friedman and Gottesman, 1983;Ito and Nakamura, 1996). However, N-mediated modification of transcribing RNA polymerase at the nutR site (between p R and t R1 ) allows transcription to efficiently proceed not only through t R1 but also through t R2 and other more distant terminators.…”

Section: Figmentioning

confidence: 99%

An RNA polymerase α subunit mutant impairs N‐dependent transcriptional antiterminationin Escherichia coli

Obuchowski

Węgrzyn

Szalewska-Pałasz

et al. 1997

Molecular Microbiology

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SummaryWe show that the rpoA341 mutation in the gene encoding the ␣ subunit of Escherichia coli RNA polymerase results in a decreased level of transcripts originating from the lytic promoters p L and p R of infecting phage. However, using lacZ fusions we demonstrate that initiation of transcription from both p L and p R is not impaired in the rpoA341 host. Rather, it is the level of the longer, antiterminated p L -and p R -derived transcripts which is altered: the activity of ␤-galactosidase in bacteria harbouring a source of N and a p L -nutL-t L1 -t I -lacZ or p R -nutR-t R1 -lacZ fusion is considerably lower in the rpoA341 mutant relative to the rpoA + strain. In the absence of the antiterminator protein N no difference is observed in the level of longer p R -derived transcripts between wild-type (rpoA + ) and mutant (rpoA341) hosts. Although synthesis of N appears to be similar in both phageinfected rpoA + and rpoA341 cells, overexpression of the N gene leads to restoration of wild-type levels of the longer p L -and p R -derived transcripts in the mutant host. While this mutation does not appear to affect vegetative phage growth in nus + backgrounds, in combination with certain nus mutations it retards lytic development. Therefore, we conclude that the rpoA341 mutation specifically interferes with the function of the N-antitermination complex, suggesting that the C-terminal domain of the RNA polymerase ␣ subunit may play an important role in N-dependent transcriptional antitermination.

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“…Reactions were carried out at 370C for 20 min, the labeled RNA products were then resolved on 4% polyacrylamide slab gels containing 8 M urea and autoradiographed as previously described (24). Galactokinase assay E. coli strain N100 containing the various recombinant plasmids was grown to logarithmic phase (OD650 = 0.6) in M56 medium with fructose as the carbon source.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro transcriptions Transcriptions were carried out in vitro using purified components as described previously (24 indicated. Reactions were carried out at 370C for 20 min, the labeled RNA products were then resolved on 4% polyacrylamide slab gels containing 8 M urea and autoradiographed as previously described (24).…”

Section: Methodsmentioning

confidence: 99%

Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

et al. 1985

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We present the nucleot'de sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions. This DNA sequence derives from the Xgal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements. We define the precise 3' junction between the bacterial and phage sequences in Xgal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage X. Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator. Lastly, we compare the gaIK genes of E. coli and the yeast S. cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.

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The relationship between function and DNA sequence in an intercistronic regulatory region in phage λ

Cited by 361 publications

References 52 publications

Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli

Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli

An RNA polymerase α subunit mutant impairs N‐dependent transcriptional antiterminationin Escherichia coli

Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

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