The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.

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Compilation and analysis of Escherichia coli promoter DNA sequences.

Cutaneous melanin pigment plays a critical role in camouflage, mimicry, social communication, and protection against harmful effects of solar radiation. Melanogenesis is under complex regulatory control by multiple agents interacting via pathways activated by receptor-dependent and -independent mechanisms, in hormonal, auto-, para-, or intracrine fashion. Because of the multidirectional nature and heterogeneous character of the melanogenesis modifying agents, its controlling factors are not organized into simple linear sequences, but they interphase instead in a multidimensional network, with extensive functional overlapping with connections arranged both in series and in parallel. The most important positive regulator of melanogenesis is the MC1 receptor with its ligands melanocortins and ACTH, whereas among the negative regulators agouti protein stands out, determining intensity of melanogenesis and also the type of melanin synthesized. Within the context of the skin as a stress organ, melanogenic activity serves as a unique molecular sensor and transducer of noxious signals and as regulator of local homeostasis. In keeping with these multiple roles, melanogenesis is controlled by a highly structured system, active since early embryogenesis and capable of superselective functional regulation that may reach down to the cellular level represented by single melanocytes. Indeed, the significance of melanogenesis extends beyond the mere assignment of a color trait.

https://www.physiology.org/doi/pdf/10.1152/physrev.00044.2003

Melanin Pigmentation in Mammalian Skin and Its Hormonal Regulation

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2009.10.036

Correlation of mRNA and protein in complex biological samples

Improved single and multicopy lac-based cloning vectors for protein and operon fusions

Correlation Between the Abundance of Escherichia Coli Transfer RNAs and the Occurrence of the Respective Codons in Its Protein Genes: A Proposal for a Synonymous Codon Choice That Is Optimal for the E. Coli Translational System

The tryptophan (trp) operon of Escherichia coli has become the basic reference structure for studies on tryptophan metabolism. Within the past five years the application of recombinant DNA and sequencing methodologies has permitted the characterization of the structural and functional elements in this gene cluster at the molecular level. In this summary report we present the complete nucleotide sequence for the five structural genes of the trp operon of E. coli together with the internal and flanking regions of regulatory information.

The complete nucleotide sequence of the tryptophan operon of Escherichia coli

Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on two structural features of the termination site--a G+C-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these two features are sufficient to specify rho-independent termination in vitro, we have introduced new sequences within a tryptophan (trp) operon structural gene to create two sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. We have compared the activity of our synthetic terminators with those of several termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. These results support a model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript.

Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro

In vivo, transcription of tryptophan (trp) operon mRNA appears to terminate at a site (trp t) 36 nucleotides after the last structural gene, and efficient function at this site requires the protein factor rho. However, distal nucleotide sequences also seem to play a role in modulating termination at trp t. We report here our in vitro studies of DNA fragments carrying portions of the trp termination region. Transcription of these DNA fragments in a purified system demonstrates that RNA polymerase actually recognizes two different termination sites. Termination at the previously characterized site, trp t, is only 25% efficient, and it is unaffected by the presence of rho factor in vitro. However, addition of rho to the transcription reaction mixture reveals that termination also occurs within a region that we have designated trp t', located about 250 bases past trp t. These two sites behave independently in vitro, whether in the tandem configuration or cloned separately, and their structural features and functional characteristics are quite different. This contrasts with the observation that termination of transcription at the end of the trp operon in vivo appears to require a rho-mediated interaction between trp t and trp t'. The possible involvement of other factors and the significance of multiple termination sites is discussed.

Tandem termination sites in the tryptophan operon of Escherichia coli

The helper dependence of satellite phage P4 superimposes an additional set of regulatory interactions on those required for the independent maintenance of P4 or its helpers. These interactions allow P4 to exploit a helper phage under a variety of circumstances and can affect expression of the immunity functions and late genes of both phages. The phage P2 lysis/lysogeny decision involves two competing repressors regulating mutually exclusive promoters in the early control region. In the absence of a helper phage, the P4 immunity function plays a role in the choice between lysogeny or the multicopy plasmid state. No evidence exists for a P4-encoded immunity repressor; in P4-lysogenic cells, expression of the P4 DNA replication gene alpha appears to be prevented by premature termination of transcription. Immunity-independent expression of alpha in the multicopy plasmid state involves initiation of transcription at an alternative upstream promoter that is positively regulated by P4 delta protein; the same promoter is activated by P2 Cox protein during derepression of P4 by P2. The mechanism of derepression of P2 by P4 remains to be determined, and the relationship between the P4 immunity and derepression functions and the mutations that allow P4 to grow with a P3 prophage helper is an intriguing area for further exploration. Expression of P2 and P4 late genes is regulated by phage-encoded, zinc-binding transcriptional activators that appear to interact directly with the alpha subunit of RNA polymerase of E. coli. Stimulation of P2 late transcription by P2 Ogr protein depends upon phage DNA replication, whereas activation of transcription from the same promoters by the related P4 delta gene product is replication-independent. Elucidation of the mechanisms underlying these interactions promises to provide new insights into strategies for control of gene expression.

Interactions between satellite bacteriophage P4 and its helpers

Background: We hypothesized that hemorrhagic shock followed by the abdominal compartment syndrome (ACS) resulted in bacterial translocation (BT) from the gastrointestinal (GI) tract. Methods: Nineteen Yorkshire swine (20-30 kg) were divided into two groups. In the experimental group, group 1 (n = 10), animals were hemorrhaged to a mean arterial pressure (MAP) of 25-30 mm Hg for a period of 30 minutes and resuscitated to baseline MAP. Subsequently, intra-abdominal pressure (IAP) was increased to 30 mm Hg above baseline by instilling sterile normal saline into the peritoneal cavity. The IAP was maintained at this level for 60 minutes. Acid/ base status, gastric mucosal ph (pHi), superior mesenteric artery (SMA) blood flow, and hemodynamic parameters were measured and recorded. Blood samples were analyzed by polymerase chain reaction (PCR) for the presence of bacteria. Spleen, lymph node, and portal venous blood cultures were obtained at 24 hours. Results were analyzed by ANOVA and are reported as mean ± SEM. The second group was the control. These animals did not have the hemorrhage, resuscitation, or intra-abdominal hypertension (IAH) but were otherwise similar to the experimental group in terms of laparotomy and measured parameters. Results: SMA blood flow in group 1 (baseline of 0.87 ± 0.10 l/min) decreased in response to hemorrhage (0.53 ± 0.10 l/min, p = 0.0001) and remained decreased with IAH (0.63 l/min ± 0.10, p = 0.0006) as compared to control and returned towards baseline (1.01 ± 0.5 l/min) on relief of IAH. pHi (baseline of 7.21 ± 0.03) was significantly decreased with hemorrhage (7.04 ± 0.03, p = 0.0003) and decreased further after IAH (6.99 ± 0.03, p = 0.0001) in group 1 compared to control, but returned toward baseline at 24 hours (7.28 ± 0.04). The mean arterial pH decreased significantly from 7.43 ± 0.01 at baseline to 7.27 ± 0.01 at its nadir within group 1 (p = 0.0001) as well as when compared to control (p = 0.0001). Base excess was also significantly decreased between groups 1 and 2 during hemorrhage (3.30 ± 0.71 vs. 0.06 ± 0.60, p = 0.001) and IAH (3.08 ± 0.71 vs. -1.17 ± 0.60, p = 0.0001). In group 1, 8 of the 10 animals had positive lymph node cultures, 2 of the 10 had positive spleen cultures, and 2 of the 10 had positive portal venous blood cultures for gram-negative enteric bacteria. Only 2 of the 10 animals had a positive PCR. In group 2, five of the nine animals had positive lymph node cultures, zero of the nine had positive spleen cultures, and one of the nine had positive portal venous blood cultures. Two of the nine animals had positive PCRs. There was no significant difference in cultures or PCR results between the two groups (Fisher's exact test, p = 0.3). Conclusion: In this study, hemorrhage followed by reperfusion and a subsequent insult of IAH caused significant GI mucosal acidosis, hypoperfusion, as well as systemic acidosis. These changes did not appear to be associated with a significant bacterial translocation as judged by PCR measurements, tissue, or blood cultures.

Gail E. Christie

Papers

The complete nucleotide sequence of the tryptophan operon of Escherichia coli

Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro

Tandem termination sites in the tryptophan operon of Escherichia coli

Interactions between satellite bacteriophage P4 and its helpers

The effects of hemodynamic shock and increased intra-abdominal pressure on bacterial translocation.