Papers by Stephen Minchin
Nucleic acids and molecular biology, 1997
E. coli RNA polymerase (RNAP) is a multisubunit enzyme with a molecular mass of nearly half a mil... more E. coli RNA polymerase (RNAP) is a multisubunit enzyme with a molecular mass of nearly half a million. The major form of RNAP in cells consists of core enzyme (subunit composition ββ′α2) in complex with the σ70 factor. σ70 is 613 amino acids in length: sequence comparisons show that it shares four regions of amino acid sequence similarity with other σ factors (regions 1, 2, 3 and 4: Fig. 1; Gross et al. 1992). It has long been known that RNAP containing σ70, is competent to initiate transcription at many promoters in the absence of any activator protein and that the σ70 subunit is essential for recognition of these promoters.
Biochemical Society Transactions, Oct 1, 2000
The sugar melibiose is one of several alternative carbon sources that E.coli can metabolise when ... more The sugar melibiose is one of several alternative carbon sources that E.coli can metabolise when glucose levels are low. The genes required for the transport and metabolism of melibiose, melB and melA respectively, are transcribed from the melAB promoter. This promoter is regulated by a transcription activator protein called MelR. MelR is a member of the AraC/XylS family of transcription activators. In previous studies, we have shown that the protein binds to several sites upstream of pmelAB. We have purified both the full-length MelR protein and its C-terminal domain (CTD). The CTD was purified using a His-tag. Both purified proteins have been used in electromobility shift assays with different D N A fragments. The CTD only binds to a subset of the sites and shows no cooperativity when binding to two sites. We postulate that cooperativity is required for MelR to bind adjacent to the transcription start site and to activate the RNA polymerase. 982 Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cp that produces an alternative isoform
Biochemical Society Transactions, Oct 1, 2000
Toxicology in Vitro, Apr 1, 2003
Hepatic gap junctional intercellular communication (GJIC), mediated principally by connexin 32, p... more Hepatic gap junctional intercellular communication (GJIC), mediated principally by connexin 32, provides a mechanism for regulating multicellular activities between neighbouring cells. The control of Cx32 gene expression at the transcriptional level has been investigated in rat liver tissue and in primary rat hepatocytes during culture. Several response elements have been identified and characterised using the electrophoretic mobility shift assay. Nuclear protein extract prepared from rat primary hepatocytes cultured for 2 h gave a larger number of DNA-protein complexes than observed with extracts from liver in vivo, including complexes containing Sp1. In contrast, nuclear extracts prepared from primary rat hepatocytes cultured for 96 h, and subject to oxidative stress, gave altered DNA-protein complexes when compared to those from hepatocytes cultured for 2 h. These results indicate that culture conditions, known to cause a loss of connexin expression, can modulate the transcription of Cx32 in hepatocytes by affecting the regulatory trans/cis-interactions of redox-sensitive zinc finger proteins within the promoter.
Journal of Bacteriology, May 15, 2000
A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymera... more A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase 70 subunit, has been used to investigate the location of 70 region 4 in different complexes at the galp 1 promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp 1 promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of 70 region 4.
European journal of biochemistry, Nov 1, 1994
Taylor & Francis, Jan 24, 2007
Essays in Biochemistry, 2019
Nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), carry genetic information ... more Nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), carry genetic information which is read in cells to make the RNA and proteins by which living things function. The well-known structure of the DNA double helix allows this information to be copied and passed on to the next generation. In this article we summarise the structure and function of nucleic acids. The article includes a historical perspective and summarises some of the early work which led to our understanding of this important molecule and how it functions; many of these pioneering scientists were awarded Nobel Prizes for their work. We explain the structure of the DNA molecule, how it is packaged into chromosomes and how it is replicated prior to cell division. We look at how the concept of the gene has developed since the term was first coined and how DNA is copied into RNA (transcription) and translated into protein (translation).
Biochemical Society Transactions, 2000
Biochemical Society Transactions, 2000
The sugar melibiose is one of several alternative carbon sources that E.coli can metabolise when ... more The sugar melibiose is one of several alternative carbon sources that E.coli can metabolise when glucose levels are low. The genes required for the transport and metabolism of melibiose, melB and melA respectively, are transcribed from the melAB promoter. This promoter is regulated by a transcription activator protein called MelR. MelR is a member of the AraC/XylS family of transcription activators. In previous studies, we have shown that the protein binds to several sites upstream of pmelAB. We have purified both the full-length MelR protein and its C-terminal domain (CTD). The CTD was purified using a His-tag. Both purified proteins have been used in electromobility shift assays with different D N A fragments. The CTD only binds to a subset of the sites and shows no cooperativity when binding to two sites. We postulate that cooperativity is required for MelR to bind adjacent to the transcription start site and to activate the RNA polymerase. 982 Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cp that produces an alternative isoform
Biochemical Journal, 1993
The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a r... more The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by c...
Brenner's Encyclopedia of Genetics, 2013
Nucleic Acids Research, 1990
Nucleic Acids Research, 2000
We have made a systematic study of how the activity of an Escherichia coli promoter is affected b... more We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the-10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the-10 and-35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions-15 and-14. Promoter activity is greatest when the 'non-template' strand carries T and G at positions-15 and-14, respectively. Promoter activity can be further enhanced by a second T and G at positions-17 and-16, respectively, immediately upstream of the first 'TG motif'. Our results show that the base sequence of the DNA segment upstream of the-10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of 'TG motifs' upstream of the promoters'-10 elements. We conclude that correctly placed 'TG motifs' are found at over 20% of E.coli promoters.
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Papers by Stephen Minchin