Abstract
Bacteriophage T4 late promoters, which consist of a simple 8-base pair TATA box, are recognized by the gene 55 protein (gp55), a small, highly diverged member of the sigma family proteins that replaces sigma(70) during the final phase of the T4 multiplication cycle. A 16-amino acid segment of gp55 that is proposed to be homologous to the sigma(70) region 2.2 has been subjected to alanine scanning and other mutagenesis. The corresponding proteins have been examined in vitro for binding to Escherichia coli RNA polymerase core enzyme and for the ability to generate accurately initiating basal as well as sliding clamp-activated T4 late transcription. Mutations in the amino acid 68-83 segment of gp55 generate a wide range of effects on these functions. The changes are interpreted in terms of the multiple steps of involvement of gp55, like other sigma proteins, in transcription. Effects of mutations on RNA polymerase core binding are consistent with the previously proposed homology of amino acids 68-82 of gp55 with sigma(70) region 2.2 and the recently determined structures of the Thermus thermophilus and Thermus aquaticus sigma(70)-RNA polymerase holoenzymes.
Highlights
All multisubunit RNA polymerases require proteins that are specialized for promoter identification and for initiation of transcription
Much of the information about the function of proteins and much of the recent key information about mechanism of action comes from the analysis of E. coli 70. 70 is a four-domain protein; each structural domain occupies a separate site on the surface of the RNA polymerase core enzyme (4 –9)
The culmination of all this effort has been the determination of structure of A holoenzymes from Thermus aquaticus (Taq) and Thermus thermophilus and of a Taq holoenzyme complex with fork junction DNA representing an open promoter complex [8, 9, 15]
Summary
Proteins—Gene 55 has been modified for this work by insertion of an N-terminal kinase tag and a C-terminal His tag into the wild type gene in expression vector pET21b. 32P-Labeled wild type gp (1.5 pmol) and 0.5 pmol of E. coli RNA polymerase core were mixed in 10 l of IP buffer (100 mM NaCl, 40 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10% (v/v) glycerol, 0.1% (v/v) Tween 20, and 266 g/ml bovine serum albumin) for 30 min in the presence or absence of 3 or 6 pmol of unlabeled mutant competitor gp in siliconized Eppendorf tubes that had been preblocked at 4 °C overnight with IP buffer. The samples, in 10 l of transcription buffer containing 10 fmol of probe DNA, 50 ng of poly(dGdC):poly(dG-dC), 200 fmol of E. coli RNA polymerase core, and 1.2 pmol of wild type or mutant gp, were incubated for 20 min at 25 °C. The Noncovalent Bond Finder Module of Protein Explorer and visual inspection was used to assess the effects of gp mutation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
