Robert G. Martin

Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli.

Transcriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator–DNA–RNA polymerase (RNP) ternary complex. Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator–DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently. We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA. In addition, we find that the ternary MarA–DNA–RNP and SoxS–DNA–RNP complexes are more stable than the corresponding Rob–DNA–RNP complex, although the binary Rob–DNA complex is often more stable than the corresponding MarA– or SoxS–DNA complexes. These results may help to explain certain puzzling aspects of the MarA/SoxS/ Rob system. We suggest that activator–RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.

Abundance and degree of dispersion of genomic d(GA) n ·d(TC) n sequences

SummaryThe abundance of d(GA)n·d(TC)n tracts was determined in genomes of rodents and primates. Dot blot hybridization assays revealed that such tracts constitute 0.40%, 0.30%, and 0.40%, respectively, of the rat, hamster, and mouse genomes, but only 0.07% and 0.05% of the human and monkey genomes. A plaque hybridization assay of rat and human genomic libraries showed that 37% and 16%, respectively, of the recombinant phages in these libraries contain d(GA)n·d(TC)n tracts. A survey of sequences stored in the GenBank data bank showed that a significant fraction of the stored rodent genes (about 2.0%) contain long d(GA)n·d(TC)n tracts (n> 30) with <10% mismatching. The primate genes contain only shorter tracts (n<15) with <10% mismatching. In addition, the rodent and the primate genes contain tracts with larger degrees of mismatching. The chicken, which represents an entirely different branch of the evolutionary tree, was found to be as low in d(GA)n·d(TC)n tracts as the primates. It is suggested that a common ancestor of the rodents has acquired the ability to amplify d(GA)n·d(TC)n tracts.

The Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40

Publisher Summary The chapter presents a simple, coherent model for transformation by SV40 that reconciles many ostensibly disparate observations. The model is consistent with the knowledge of normal mammalian DNA replication. The model also accounts for a number of observations associated with induction of host DNA replication by SV40 and with transformation by the virus. The hypothesis is that transformation by SV40 unlike PyV is largely explicable in terms of the ability of the 90K T-antigen to initiate DNA synthesis. The enzymatic activity associated with the initiation of DNA synthesis, be it endonucleolytic attack, transcription, topoisomerization, or any other activity; all of the data are consistent with the notion that the 90K T-antigen can substitute for the normal host initiator function. By bypassing the normal control for initiation of DNA synthesis, the T-antigen can propel the cell into rounds of replication beyond those exhibited by non-transformed cells in the same conditions. The interaction of T-antigen with host DNA might also account for gene activation through a direct effect on transcription. The rate of T-antigen synthesis is controlled partly by the host, possibly as a function of the site of integration of the viral genome into that of the host. The activity of the 90K t-antigen is also controlled partly by host modification. The 20K t-antigen plays a role in the establishment of transformation of resting cells in tissue culture. It may also have some role in maintenance of the transformed phenotype in tissue culture, but if so, its role can be supplanted by normal sera.

Ribonucleotide composition of the genetic code.

Abstract Randomly-mixed polynucleotides were found to direct the incorporation of different amino acids into protein. Using this technique the ribonucleotide composition of the RNA code corresponding to fifteen amino acids was determined. A minimum coding ratio of three nucleotides per amino acid was demonstrated. Two coding units corresponding to leucine were found; thus a part of the code was shown to be degenerate.

Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration.

The paralogous transcriptional activators MarA, SoxS, and Rob activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and by measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between the MarA concentration needed for half-maximal promoter activity in vivo and marbox binding affinity in vitro was poor; and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS, implying that the two activators interact with RNA polymerase in different ways at the different promoters. Thus, the concentration and nature of activator determine which regulon promoters are activated, as well as the extent of their activation.

Transcriptional and translational regulation of the marRAB multiple antibiotic resistance operon in Escherichia coli

The marRAB multiple antibiotic resistance operon of Escherichia coli is autorepressed by MarR. MarR binds to two palindromic sequences in vitro: site I lies between and overlaps the −35 and −10 hexamers for RNA polymerase binding; site II lies between the transcription start site and the GTG initiation codon of marR. To assess the importance of these sites in vivo, the effects of mutant sites on transcription were analysed using fusions to lacZ in the presence and absence of wild‐type MarR. When both sites were wild type, transcription in the derepressed marR‐deleted strain was 19‐fold that of the wild‐type strain; when only site I or site II was wild type, this ratio was reduced to 4.3‐ and 2.6‐fold, respectively, showing that full repression requires both sites, but some repression can occur at one site independently of the other. Translational fusions of the wild‐type promoter to lacZ demonstrated that marR translation proceeds at only 4.5% of the transcription rate. Analysis of translational fusions with mutant leader sequences demonstrated that the principal reason for inefficient translation is a weak Shine–Dalgarno (SD) sequence, AGG(G). Although the SD sequence is located within the potential stem–loop structure of site II, no evidence for occlusion of the SD sequence was found in the wild‐type strain. However, a single basepair mutation that strengthens the stem–loop structure drastically reduced the translational efficiency. Substitution of ATG for GTG as the initiation codon increased translational efficiency by 50%. Increasing the 5u2003bp spacing between the SD sequence and the GTG codon by one to four bases reduced the translational efficiency by 50–75%. Inefficient translation of marR may help to sensitize the cell to environmental signals.

Reduction of Cellular Stress by TolC-Dependent Efflux Pumps in Escherichia coli Indicated by BaeSR and CpxARP Activation of spy in Efflux Mutants

Escherichia coli has nine inner membrane efflux pumps which complex with the outer membrane protein TolC and cognate membrane fusion proteins to form tripartite transperiplasmic pumps with diverse functions, including the expulsion of antibiotics. We recently observed that tolC mutants have elevated activities for three stress response regulators, MarA, SoxS, and Rob, and we suggested that TolC-dependent efflux is required to prevent the accumulation of stressful cellular metabolites. Here, we used spy::lacZ fusions to show that two systems for sensing/repairing extracytoplasmic stress, BaeRS and CpxARP, are activated in the absence of TolC-dependent efflux. In either tolC mutants or bacteria with mutations in the genes for four TolC-dependent efflux pumps, spy expression was increased 6- to 8-fold. spy encodes a periplasmic chaperone regulated by the BaeRS and CpxARP stress response systems. The overexpression of spy in tolC or multiple efflux pump mutants also depended on these systems. spy overexpression was not due to acetate, ethanol, or indole accumulation, since external acetate had only a minor effect on wild-type cells, ethanol had a large effect that was not CpxA dependent, and a tolC tnaA mutant which cannot accumulate internal indole overexpressed spy. We propose that, unless TolC-dependent pumps excrete certain metabolites, the metabolites accumulate and activate at least five different stress response systems.

Transcriptional activation by MarA, SoxS and Rob of two tolC promoters using one binding site: a complex promoter configuration for tolC in Escherichia coli

The Escherichia coli tolC encodes a major outer membrane protein with multiple functions in export (e.g. diverse xenobiotics, haemolysin) and as an attachment site for phage and colicins. tolC is regulated in part by MarA, SoxS and Rob, three paralogous transcriptional activators which bind a sequence called the marbox and which activate multiple antibiotic and superoxide resistance functions. Two previously identified tolC promoters, p1 and p2, are not regulated by MarA, SoxS or Rob but p2 is activated by EvgAS and PhoPQ which also regulate other functions. Using transcriptional fusions and primer extension assays, we show here that tolC has two additional strong overlapping promoters, p3 and p4, which are downstream of p1, p2 and the marbox and are activated by MarA, SoxS and Rob. p3 and p4 are configured so that a single marbox suffices to activate transcription from both promoters. At the p3 promoter, the marbox is separated by 20 bp from the −10 hexamer for RNA polymerase but at the p4 promoter, the same marbox is separated by 30 bp from the −10 hexamer. The multiple tolC promoters may allow the cell to respond to diverse environments by co‐ordinating tolC transcription with other appropriate functions.

Sequence similarities among monkey ori-enriched (ors) fragments

Nucleotide sequences have been determined for eight ors (ori-enriched sequence) fragments isolated from monkey DNA by a method that was designed to enrich for origins of DNA replication [Kaufmann et al., Mol. Cell. Biol. 5 (1985) 721-727]. Evidence has been presented that some or possibly all of these sequences can serve, albeit inefficiently, as oris in vivo [Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA 84 (1987) 6668-6672]. Two of the fragments were found to contain the long terminal repeat-like elements of the O-family of moderately repetitive sequences that are present in human DNA as a transposon-like element [Paulson et al., Nature 315 (1985) 359-361]. Extensive pair-wise comparisons of the sequences failed to detect any statistically significant common sequences, except for long asymmetrically distributed A + T-rich stretches. Nonetheless, when the ors fragments were examined for the presence of published consensus sequences, seven of eight were found to contain the control sequence described by Dierks et al. [Cell 32 (1983) 695-706], and the same seven of eight were found to contain both the scaffold attachment region T consensus [Gasser and Laemmli, Cell 46 (1986) 521-530] and the minimal Saccharomyces cerevisiae autonomously replicating sequence consensus [e.g., Palzkill and Newlon, Cell 53 (1988) 441-450].

Versatility of the carboxy-terminal domain of the α subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA

The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon. Here, we identify the interacting surfaces of MarA and of the carboxy‐terminal domain of the α subunit of RNAP (α‐CTD) by NMR‐based chemical shift mapping. Spectral changes were monitored for a MarA‐DNA complex upon titration with α‐CTD, and for α‐CTD upon titration with MarA‐DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that α‐CTD uses a ‘265‐like determinant’ to contact MarA at a surface distant from the DNA. This is unlike the interaction of α‐CTD with the CRP or Fis activators where the ‘265 determinant’ contacts DNA while another surface of the same α‐CTD molecule contacts the activator. These results reveal a new versatility for α‐CTD in transcriptional activation.