Richard R. Burgess

Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes.

An improved method is described for the renaturation of microgram amounts of proteins from sodium dodecyl sulfate-polyacrylamide gels. The protein band is visualized in the gel by KCl staining, the band cut out and crushed, and the protein eluted by diffusion in a buffer containing 0.1% sodium dodecyl sulfate. Protein is concentrated and sodium dodecyl sulfate is removed by acetone precipitation of the sample. Renaturation of the protein occurs after the precipitate is dissolved in 6 m guanidine hydrochloride and then diluted. The activity of the sigma subunit of Escherichia coli RNA polymerase can be recovered with 98–100% efficiency after electrophoresis in an SDS-gel and renaturation by this technique. To assess whether the method is generally applicable we tested some or all of the steps involved in the procedure using E. coli transcription termination factor rho, β-galactosidase, alkaline phosphatase, wheat α-amylase, and DNA topoisomerase. We show how the method can be used to determine the approximate molecular weight of the DNA topoisomerase polypeptide by sectioning a gel on which a partially pure sample has been fractionated by electrophoresis.

The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression

The codon preference plot is useful for locating genes in sequenced DNA, predicting the relative level of their expression and for detecting DNA sequencing errors resulting in the insertion or deletion of bases within a coding sequence. The three possible reading frames are displayed in parallel along with the open reading frames and plots of the location of rare codons in each reading frame.

Promoter recognition and discrimination by EsigmaS RNA polymerase.

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing σS (EσS), and it is known that there is some overlap between the promoters recognized by EσS and by the major E. coli holoenzyme (Eσ70), the sequence elements responsible for promoter recognition by EσS are not well understood. To define the DNA sequences recognized best by EσSin vitro, we started with random DNA and enriched for EσS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EσS contained the known consensus elements (−10 and −35 hexamers) for recognition by Eσ70. Using genetic and biochemical approaches, we show that EσS and Eσ70 do not achieve specificity through ‘best fit’ to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EσS‐specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Eσ70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EσS versus Eσ70 thus presents an alternative solution to the problem of promoter selectivity.

Refolding solubilized inclusion body proteins.

The vast majority of protein purification is now done with cloned, recombinant proteins expressed in a suitable host. The predominant host is Escherichia coli. Many, if not most, expressed proteins are found in an insoluble form called an inclusion body (IB). Since the target protein is often relatively pure in a washed IB, the challenge is not so much to purify the target, but rather to solubilize an IB and refold the protein into its native structure, regaining full biological activity. While many of the operations of this process are quite general (expression, cell disruption, IB isolation and washing, and IB solubilization), the precise conditions that give efficient refolding differ for each protein. This chapter describes the main techniques and strategies for achieving successful refolding.

Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase

We have constructed a plasmid that overexpresses 100-fold the sigma subunit of Escherichia coli RNA polymerase. The plasmid was constructed by placing the pLoL promoter-operator of bacteriophage lambda upstream from rpoD, the gene encoding the sigma subunit. A simple procedure for purification of the overexpressed protein has been developed based on guanidine hydrochloride denaturation/renaturation, DEAE cellulose chromatography, and Sephacryl S-200 chromatography. The purified product has been characterized and found to be indistinguishable from normally expressed sigma protein purified by previous protocols as judged by enzymatic activity, heat inactivation, and partial proteolysis.

Localization of a ς70 Binding Site on the N Terminus of the Escherichia coli RNA Polymerase β′ Subunit

The Escherichia coli genome encodes genes for seven different ς subunit species while only having single genes for the α, β, and β′ subunits that make up the RNA polymerase core enzyme. The various ς factors compete for binding to the core enzyme, upon which they confer promoter DNA-specific transcription initiation to the polymerase. We have mapped a major interaction site between one of the ς species, ς70, and β′. Using far-Western blotting analysis of chemically cleaved and genetically engineered protein fragments, we have identified a N-terminal fragment of β′ (residues 60–309) that could bind ς70. We were able to more precisely map the interaction domain to amino acid residues 260–309 of β′ using nickel nitrilotriacetic acid co-immobilization assays.

Manganese, Mn-dependent peroxidases, and the biodegradation of lignin

Manganese and Mn-dependent peroxidases have been implicated in the enzymatic degradation of lignin. However, the specific role of manganese is uncertain. We report here the novel observation that in the absence of enzyme, suitably chelated Mn3+ is a ligninolytic agent capable of oxidizing veratryl alcohol, lignin model compounds, and lignin. We also demonstrate the unexpected effect of reducing agents which stimulate the oxidations by Mn3+. The stimulation is apparently through the production of a reduced oxygen species likely to be superoxide. These observations provide a fresh insight into the process of lignin biodegradation.

Stringent response in Escherichia coli induces expression of heat shock proteins.

The rpoD gene (encoding the 70,000 Mr sigma subunit of Escherichia coli RNA polymerase) is the most distal gene in an operon that contains three genes. The promoter-proximal gene is rpsU (encoding ribosomal protein S21) and the middle gene is dnaG (encoding DNA primase). During the stringent response, caused by a deficiency in an aminoacyl-tRNA, expression of rpsU is decreased, while expression of rpoD is not. This disco-ordinate regulation is due to increased transcription from a minor promoter upstream from rpoD, in the dnaG gene. Transcription from this promoter is also increased during the heat shock response. Expression of other heat shock proteins was found to increase during the stringent response. Thus, the stringent response in E. coli induces expression of heat shock proteins. The requirements for this stringent induction of the heat shock proteins differ from those for temperature induction during the heat shock response.

Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase

The rpoD gene encoding the sigma subunit of E. coli RNA polymerase is cotranscribed with rpsU and dnaG, encoding ribosomal protein S21 and DNA primase, respectively. After temperature upshift, a heat shock promoter (Phs) located within dnaG is transiently induced, causing increased transcription of rpoD. The extent of induction is sufficient to account for the heat shock response of sigma synthesis. The initiation site of this promoter was located about 360 bp upstream of rpoD by promoter cloning and S1 nuclease mapping. Plasmid deletions generated with Bal 31 nuclease show that the DNA sequence CTGCCACCC in the -44 to -36 region of this promoter is necessary for its heat shock activity. Heat induction of transcription from Phs is under the control of HtpR, a positive regulator of the heat shock response.

A Coiled-Coil from the RNA Polymerase β′ Subunit Allosterically Induces Selective Nontemplate Strand Binding by σ70

Abstract For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the −10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the β′ subunit (aa 262–309) induces σ 70 to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the β′ coiled-coil and region 2.2 of σ 70 promotes an allosteric transition that allows σ 70 to selectively recognize the nontemplate strand. As the β′ 262–309 peptide can function with the previously crystallized portion of σ 70 , nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.