PubMed Health⌕ Search

Biomedical subjects

S Busby

Publications and source records attributed to S Busby.

At least 19 recordsLinked to original sources
PubMed2001

Activation and repression of transcription initiation in bacteria.

Transcription initiation is the principal step at which bacterial gene expression is regulated. Bacterial transcription is due to a single multisubunit RNA polymerase. The potential transcription initiation rate of any promoter is set by the efficiency with which RNA polymerase recognizes the different promoter sequence elements. The sigma subunit plays the major role in the process of promoter recognition. Different RNA polymerase sigma subunits can guide RNA polymerase to different promoters. The E. coli genome encodes seven different sigma subunits, each of which allows the cell to respond to different environmental stimuli. A large number of transcription factors up-regulate and down-regulate expression from different promoters in response to environmental signals. Many transcription activators function by making a direct interaction with RNA polymerase. Some activators function by altering the conformation of promoter DNA. Most transcription repressors function by blocking access of RNA polymerase to their target promoter. In some cases, optimal repression depends on multiply bound repressor molecules that interact in complex ways. Many promoters are regulated by more than one transcription factor. A variety of mechanisms whereby a promoter can be regulated by a repressor and an activator, or by two activators, is known.

Bacteria↗
PubMed2000

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study.

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.

Base Sequence↗
PubMed1999

Transcription activation by catabolite activator protein (CAP).

Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.

Cyclic AMP Receptor Protein↗
PubMed1998

Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase sigma70 subunit.

The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation.

Alanine↗
PubMed1998

Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm.

During transcription activation at FNR-dependent promoters where the DNA site for FNR overlaps the -35 element, a surface-exposed activating region in the upstream subunit of the FNR dimer interacts with the C-terminal domain of the RNA polymerase alpha subunit. Starting with a cloned fnr gene encoding a defective FNR derivative carrying substitutions in this activating region, we screened a library of random mutations to identify substitutions that restored FNR activity. Activity can be restored by substitutions at residues T118, E47 and K60. The locations of these residues identify three separate surface-exposed regions of FNR that can play a role in transcription activation. These three regions appear to be analogues of Activating Region 1, Activating Region 2 and Activating Region 3 of the cyclic AMP receptor protein, CRP: our results underscore the similarities between FNR and CRP.

Bacterial Proteins↗
PubMed1998

Oviposition and incubation environmental effects on embryonic diapause in a ground cricket

Maternal effects on offspring phenotype are well known in organisms inhabiting seasonal environments. Mothers that perceive climatological changes indicative of winter's onset will often produce increasing numbers of offspring that enter a state of arrested morphogenesis known as 'diapause'. In this study of bivoltine Allonemobius socius ground crickets, we manipulated the abiotic environment experienced by ovipositing females (i.e. maternal-oviposition environment) and that experienced by offspring incubating as eggs (i.e. egg-incubation environment) to assess the degree to which mothers contribute to the expression of embryonic diapause in her offspring. Analysis of variance components indicated that variation in maternal-oviposition environment contributed only about 4% to the total variation in diapause incidence, compared to about 24% from embryos responding directly to variation in their incubation environment. Moreover, the 8% contribution from between-family variation was significant, suggesting that parental genes and maternal biotic and abiotic environments contribute to diapause expression in offspring. Although these findings suggest that maternal physi-ology itself contributes little to embryonic diapause variation in A. socius, they do not preclude other maternal behaviours (e.g. placement of eggs at different soil depths) that may affect offspring diapause.Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1997

Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein.

The Escherichia coli nir and nrf operons, which encode alternative nitrite reductases expressed during anaerobic growth, are subject to catabolite regulation. Transcription from the nir promoter is maximal when bacteria are grown in rich media such as Lennox broth supplemented with glucose. Conversely, expression of the nrf operon is suppressed by rich media, but stimulated during growth in minimal medium with glycerol and fumarate. The role of the catabolite repressor-activator (Cra) protein in catabolite regulation of the nir and nrf promoters was investigated. Transcription from the nir promoter was repressed by Cra when cells were grown in minimal medium with glycerol and fumarate. Crude protein extracts from a strain overproducing Cra encoded on a multicopy plasmid retarded a nir promoter fragment in a mobility shift assay, confirming that the observed Cra-dependent repression was due to the direct interaction of Cra with the regulatory region of the nir operon. Furthermore, the inclusion of fructose 1-phosphate, an effector of Cra DNA-binding activity, in the assay decreased the ability of Cra to retard the nir promoter fragment. In contrast, transcription from the nrf promoter was not regulated by Cra under any of the growth conditions tested.

Bacterial Proteins↗
PubMed1997

Transcription activation at class II CAP-dependent promoters.

Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP-dependent promoters, the DNA target site for CAP is centred near position -42, overlapping and replacing the -35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is 'anti-inhibition,' overcoming an inhibitory effect of the RNAP alpha subunit C-terminal domain (alpha CTD). This component involves direct contact between amino acids 156-164 (activating region 1) of the upstream subunit of the CAP dimer and a target in alpha CTD. The second component is 'direct activation', facilitating isomerization of the RNAP-promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP alpha subunit N-terminal domain (alpha NTD).

DNA-Directed RNA Polymerases↗
PubMed1996

The role of the genes nrf EFG and ccmFH in cytochrome c biosynthesis in Escherichia coli.

It has been suggested that two groups of Escherichia coli genes, the ccm genes located in the 47-min region and the nrfEFG genes in the 92-min region of the chromosome, are involved in cytochrome c biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochrome c synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochrome c assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of the c-type cytochromes in E. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochrome C550 and cytochrome C552, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least two c-type cytochromes, cytochrome C552 and NrfB, but NrfF is not essential for this pathway. Genes similar to nrfE, nrfF and nrfG are present in the E. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of all c-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochrome c biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haem c to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.

Cytochrome c Group↗
PubMed1996

Location of essential sequence elements at the Escherichia coli melAB promoter.

The Escherichia coli melAB promoter has been cloned on a short DNA fragment and subjected to deletion mutagenesis, random mutagenesis and site-directed mutagenesis. In previous work we had shown that expression from the melAB promoter is triggered by melibiose and that this requires the MelR transcription activator. Melibiose-dependent expression is suppressed by deletions that remove both DNA-binding sites for MelR and by point mutations in the -10 hexamer, the -35 hexamer and the region just upstream of the -35 hexamer. The point mutations identify promoter elements that are essential for triggering the melAB promoter. The importance of these elements was confirmed by site-directed mutagenesis. The results show that the organization of the melAB promoter is fundamentally different from the organization of other bacterial promoters controlled by homologues of MelR.

Base Sequence↗
PubMed1996

Protein-protein interactions during transcription activation: the case of the Escherichia coli cyclic AMP receptor protein.

The Escherichia coli cyclic AMP receptor protein (CRP) is a homodimeric transcription activator triggered by cyclic AMP. Escherichia coli contains more than 100 different promoters that can be activated by CRP: in most cases the CRP acts by making direct contact with RNA polymerase. Remarkably, there is considerable variation in the location of the DNA site for CRP from one CRP-dependent promoter to another. Genetic methods have been used to locate the activating regions of CRP that make contact with RNA polymerase at promoters of different architectures. At promoters where the DNA site for CRP is centred near to positions -61, -71 or -81 (i.e. 61, 71 or 81 base pairs upstream of the transcript start-point, respectively), a single surface-exposed loop (Activating Region 1) in the downstream subunit of the CRP dimer makes contact with RNA polymerase. The contact site in RNA polymerase is located in one of the C-terminal domains of two RNA polymerase alpha subunits. At promoters where the DNA site for CRP is centred near to position-41, both subunits of the CRP dimer make contact with RNA polymerase via three separate surface exposed regions (Activating Regions 1, 2 and 3). At these promoters, where bound CRP overlaps with RNA polymerase-binding elements, the C-terminal domains of the polymerase alpha subunits are displaced and bind upstream of CRP. Activation at a number of E. coli promoters is dependent on binding of two CRP dimers, with one dimer bound near to position-41 and the other dimer bound further upstream. In these cases, both bound CRP dimers contact RNA polymerase. The CRP dimer bound around position-41 contacts RNA polymerase via Activating Regions 1, 2 and 3, whereas the upstream bound CRP dimer contacts one of the displaced alpha C-terminal domains via Activating Region 1 in the downstream CRP subunit. Thus in these cases, codependence on two activators is due to simultaneous contacts between separate activators and RNA polymerase. This mechanism allows great flexibility, as any activator that can contact the C-terminal domain of the RNA polymerase alpha subunits can act cooperatively with CRP.

Binding Sites↗
PubMed1995

The Escherichia coli RNA polymerase alpha subunit: structure and function.

Recent work has established that the Escherichia coli RNA polymerase alpha subunit consists of an amino-terminal domain containing determinants for interaction with the remainder of RNA polymerase, a carboxy-terminal domain containing determinants for interaction with DNA and interaction with transcriptional activator proteins, and a 13-36 amino acid unstructured and/or flexible linker. These findings suggest a simple, integrated model for the mechanism of involvement of alpha in promoter recognition and transcriptional activation.

Base Sequence↗
PubMed1995

Transcriptional co-activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem.

Previous work with semi-synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around -91 bp. The ansB promoter in Escherichia coli is co-activated in a co-dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around -91 and -41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co-activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co-dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co-activate when located upstream at around -91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co-dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co-dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co-dependence is determined by the nature of the activator plus the promoter context.

Asparaginase↗