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. 2000 Jan;182(1):221-4.
doi: 10.1128/JB.182.1.221-224.2000.

Effects of amino acid substitutions at conserved and acidic residues within region 1.1 of Escherichia coli sigma(70)

C W Bowers  1 , A McCracken, A J Dombroski
Affiliations

Effects of amino acid substitutions at conserved and acidic residues within region 1.1 of Escherichia coli sigma(70)

C W Bowers et al. J Bacteriol. 2000 Jan.

Abstract

Amino acid substitutions in Escherichia coli sigma(70) were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.

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Figures

FIG. 1
FIG. 1
Run-off transcription analysis. (A) A representative run-off transcription time course experiment is shown for Eς70 and ς70 mutants at 37°C. Each experiment was repeated at least five times, and the error was less than 15%. (B) Abortive transcription by Eς70D61A at 37°C. Synthesis of the three nucleotide abortive RNA transcripts (ApUpG) from λ pR is shown relative to the wild type. The holoenzyme concentration was 0.008 μM and the template concentration was 1.5 nM. (C) Run-off transcription as a function of temperature. A comparison of run-off transcription activities at 60 min at 25 and 42°C is shown. Activities are normalized to the wild type, and the error indicated was less than 15%. WT, wild type.
FIG. 1
FIG. 1
Run-off transcription analysis. (A) A representative run-off transcription time course experiment is shown for Eς70 and ς70 mutants at 37°C. Each experiment was repeated at least five times, and the error was less than 15%. (B) Abortive transcription by Eς70D61A at 37°C. Synthesis of the three nucleotide abortive RNA transcripts (ApUpG) from λ pR is shown relative to the wild type. The holoenzyme concentration was 0.008 μM and the template concentration was 1.5 nM. (C) Run-off transcription as a function of temperature. A comparison of run-off transcription activities at 60 min at 25 and 42°C is shown. Activities are normalized to the wild type, and the error indicated was less than 15%. WT, wild type.
FIG. 1
FIG. 1
Run-off transcription analysis. (A) A representative run-off transcription time course experiment is shown for Eς70 and ς70 mutants at 37°C. Each experiment was repeated at least five times, and the error was less than 15%. (B) Abortive transcription by Eς70D61A at 37°C. Synthesis of the three nucleotide abortive RNA transcripts (ApUpG) from λ pR is shown relative to the wild type. The holoenzyme concentration was 0.008 μM and the template concentration was 1.5 nM. (C) Run-off transcription as a function of temperature. A comparison of run-off transcription activities at 60 min at 25 and 42°C is shown. Activities are normalized to the wild type, and the error indicated was less than 15%. WT, wild type.
FIG. 2
FIG. 2
Stability of ς70D61A in vitro and in vivo. (A) Limited trypsinolysis. ς70D61A (2 μg) was subjected to trypsin digestion at three temperatures, as indicated, to assess possible structural defects. Wedges indicate increasing trypsin concentration (0.0125, 0.0625, and 0.025 μg). Digestion of wild-type (WT) ς70 is shown for reference in the top panel. Fragments were resolved on a sodium dodecyl sulfate–8% polyacrylamide gel and visualized with Coomassie brilliant blue staining. (B) Immunoblot analysis of culture lysates of strain 19284 (wild-type [WT] ς70 and ς70D61A). Exponentially growing cells at 37°C were upshifted to 44°C. Lysates were prepared from cells harvested at the indicated time points following temperature upshift, and proteins were resolved on a sodium dodecyl sulfate–8% polyacrylamide gel. Following Western transfer, histidine-tagged ς70 proteins were detected by using a six-His-tagged monoclonal antibody (Clontech).
FIG. 3
FIG. 3
Stability of initiated complexes. Nitrocellulose filter retention under low (0.1 M NaCl)- and high (0.8 M NaCl)-stringency wash conditions is shown. The fraction of mutant complexes retained is normalized to the fraction of wild-type (WT) complexes retained after allowing formation for 2 and 30 min following mixing of RNA polymerase with DNA. Amino acid substitutions are indicated.
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