Polymerase Recognition Sequence Research Articles

A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in Salmonella

BackgroundBacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined.ResultsHere, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation.ConclusionsWe identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system.

NF90 Regulates Cell Cycle Exit and Terminal Myogenic Differentiation by Direct Binding to the 3′-Untranslated Region of MyoD and p21WAF1/CIP1 mRNAs

NF90 and splice variant NF110/ILF3/NFAR are double-stranded RNA-binding proteins that regulate gene expression. Mice with targeted disruption of NF90 were engineered. NF90(-/-) mice were born small and weak and succumbed to perinatal death within 12 h because of neuromuscular respiratory failure. Lung inflation and morphology were normal in NF90(-/-) mice. The diaphragm and other skeletal muscles in NF90(-/-) mice demonstrated disorganized arrangement and paucity of myofibers, evidence of myocyte degeneration and increased apoptosis. The expression of myogenic regulators, MyoD, myogenin, and p21WAF1/CIP1, was severely decreased in NF90(-/-) mice. These myogenic transcription factors and cell cycle inhibitors are regulated in part through post-transcriptional mRNA stabilization. Northwestern blotting revealed that NF90 is the principal and specific p21WAF1/CIP1 and MyoD 3'-untranslated region RNA-binding protein in developing skeletal muscles. NF90 regulates transcription factors and a cell cycle inhibitor essential for skeletal muscle differentiation and for survival.

Frankia sequences exhibiting RNA polymerase promoter activity.

Frankia are Gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen either in the free-living state or in symbiosis with a variety of woody plants. Only a few Frankia genes have been sequenced and gene expression is not well characterized. To isolate a segment of Frankia DNA that functions as an RNA polymerase promoter, fragments of Frankia strain ArI5 genomic DNA were cloned upstream of a promoterless, Vibrio harveyi luxAB cassette. Constructs were screened for luminescence in E. coli and positive clones assayed for in vitro transcription activity with a partially purified Frankia RNA polymerase extract. Primer extension analysis of in vitro transcripts produced from one clone, GLO7, identified two major transcription start sites, TSP-1 and TSP-2, 52 bp apart. Deletion analysis then localized sequences essential for promoter activity. The upstream promoter region, GLO7p1, contains sequences resembling the -35 element of a Streptomyces promoter and the -35 and -10 elements of the canonical E. coli promoter. Also within this region are two pentamers identical to sequences near the 5' end of the Frankia strain CpI1 glutamine synthetase gene. The second promoter, GLO7p2, contains a putative NtrC binding site at -145 and a possible sigma(N)-RNA polymerase recognition sequence at -14 suggesting that GLO7p2 may be a nitrogen-regulated promoter. An in vivo transcript representing an ORF of 498 aa starting 64 bp downstream of the distal transcription start, TSP-1, was detected by RT-PCR. This supports the conclusion that this DNA fragment has promoter activity in vivo as well as in vitro.

The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro.

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.

Integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter Pu of Pseudomonas putida.

In the presence of m-xylene, the Pu promoter of the TOL plasmid of Pseudomonas putida is activated by the prokaryotic enhancer-binding protein XylR. The intervening DNA segment between the upstream activating sequences (UASs) and those for RNA polymerase binding contains an integration host factor (IHF) attachment site that is required for full transcriptional activity. In the absence of IHF, the Pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory proteins. Such illegitimate activation does not require the binding of the heterologous regulators to DNA and it is suppressed by bent DNA structures, either static or protein induced, between the promoter core elements (UAS and RNA polymerase recognition sequence). The role of IHF in some sigma 54 promoters is, therefore, not only a structural aid for assembling a correct promoter geometry but also that of an active suppressor (restrictor) of promiscuous activation by heterologous regulators for increased promoter specificity.

Regulation of Mitochondrial Gene Expression in Saccharomyces cerevisiae

This chapter focuses on the regulation of mitochondrial gene expression in yeast (Saccharomyces cerevisiae), and tries to explain how these gene expression is regulated. The chapter comprises the newer information on nuclearly encoded factors that affect mitochondrial transcription; mRNA processing, stability, and translation; and posttranslational modification and assembly. Transcription of yeast mitochondrial operons appears to be somewhat more complicated than transcription of phage genes but less complex than E. coli transcription. The 9-bp mitochondrial promoter is positioned immediately upstream of the start of transcription, which is reminiscent of the spacing of the polymerase recognition sequence in T3 and T7 phage genes. Though the mitochondrial enzyme contains two subunits, the catalytic subunit RP041 has considerable sequence similarity to the single subunit T-odd polymerases. With the exception of one transcript, that for a second tRNAthr, mitochondrial transcription units are multigenic. In some cases the initial transcripts contain multiple tRNAs, in other cases combinations of mRNAs and tRNAs or rRNAs and tRNAs. The RNAs involved in mitochondrial translation are all encoded on the mitochondrial genome, including the large and small ribosomal RNAs and the tRNAs.

Spo0A controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE.

The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis. Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by sigma A-associated RNA polymerase (E sigma A), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17- or 18-bp spacing typical of promoters utilized by E sigma A. Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence. The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by E sigma A. Similar suppression experiments failed to provide evidence for a direct interaction between E sigma A and the "-35-like" element, however, and DNase I protection experiments suggested instead that the Spo0A protein binds to a site overlapping this -35-like hexamer. Moreover, the effects of mutations within the -35-like hexamer on the binding of Spo0A in vitro paralleled their effects on transcription in vivo. We suggest that spoIIE belongs to a class of early-intermediate sporulation genes whose transcription by E sigma A is activated by the Spo0A protein.

44] Cloning of complementary DNA inserts from phage DNA directly into plasmid vector

Publisher Summary The desired complementary DNA (cDNA) inserts are usually sub cloned from the positive phages into appropriate plasmid or M13 vectors for large-scale production. The cDNA insert isolated from the subcloning vector is then subjected to restriction enzyme mapping, southern blotting and hybridization, or nucleotide sequencing. Several methods have been designed to analyze the cDNA insert directly from the phage clone. Double-stranded h DNA can serve as a template for sequencing, using the chain termination method. The cDNA insert can be amplified for subcloning by the PCR. By incorporating the T7 RNA polymerase recognition sequence and the translation initiation codon into a primer, the amplification by the PCR has allowed subsequent in vitro transcription and translation of the phage cDNA inserts into protein. The method is likely to be extended to the cloning of inserts from other λ vectors when bacteria host contains pMC9— that is, (Y1088, Y1089, or YI090) or other plasmid vectors. More importantly, combination of this method with the PCR method, which allows the analysis of DNA directly from single bacterial colonies by boiling in H2O, made it practical to clone several cDNA inserts simultaneously.

Mapping of the puh Messenger RNAs from Rhodospirillum rubrum: Evidence for tandem promoters

Abstract The mRNA transcripts of Rhodospirillum rubrum gene puh, coding for the H subunit of the photoreaction center, and of genes flanking puh were analyzed by blot hybridization. Open reading frame G115, upstream of structural gene puh, is transcribed as a 2.25-kilobase mRNA. Gene puh itself is transcribed as two mRNAs of 1118 and 1032 nucleotides. Mung bean nuclease protection analysis shows that the puh transcripts have different 5' termini within open reading frame G115 and a unique rho-independent termination signal within open reading frame I2372. The lifetimes of the puh messages, as determined by an oxygen blockade of transcription, were 10 and 12 min for the large and small puh mRNAs, respectively. An expression vector carrying a chloramphenicol acetyltransferase gene was used to select promoters in DNA stretches upstream of the startpoints of each of these transcripts. Chloramphenicol resistance was expressed in Escherichia coli, using as a promoter a 179-nucleotide stretch upstream of the small mRNA startpoint but not from a 124-nucleotide stretch upstream of the large mRNA startpoint. The promoter for the small mRNA, designated Ppuh2, is thought to encompass in its -10 and -35 regions a sigma 70-like RNA polymerase recognition sequence. The region upstream of the large message startpoint contains a sequence similar in its -12 and -24 regions to promoter sequences recognized by the sigma 60 RNA polymerase holoenzyme. This is designated as promoter Ppuh1.Ppuh1 is proposed to be strictly regulated by light intensity and by oxygen tension while Ppuh2 would be less sensitive to these parameters.

Mutations in the RNA polymerase recognition sequence of theKlebsiella pneumoniae nifHpromoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences

Positive control of the wild-type Klebsiella pneumoniae nifH promoter by the NifA protein requires that NifA is bound at the upstream activator sequence (UAS). By introducing base substitutions at -15 to -17 in the RNA polymerase recognition sequence of the nifH promoter, positive control by a form of NifA unable to bind to the UAS was greatly increased when compared to the wild-type promoter. Transcriptional activation still required the rpoN encoded sigma factor and was initiated at the same nucleotide as in the wild-type promoter. Mutations at -15 to -17 suppressed the requirement that the UAS should be located on the correct face of the DNA helix with respect to the RNA polymerase recognition sequence in order that titration of NifA and efficient activation occur. This result supports the suggestion that upstream bound NifA interacts with the RNA polymerase-RpoN complex. To examine the minimal carboxy terminal sequences required for the positive control function of NifA a series of carboxy terminal deletions were constructed. Efficient positive control at a UAS-independent promoter was only observed in deletions which did not extend beyond the proposed boundary separating the carboxy terminal NifA DNA-binding domain from its central domain.

Multiple mRNAs are generated from the chicken lysozyme gene

We have determined the DNA sequence of a 770 by Pst I fragment containing 450 nucleotides of the 5′ flanking region of the chicken lysozyme gene. S1-nuclease mapping was performed to localize the 5′ end of nuclear RNA containing lysozyme-specific sequences and of the mRNA. We present evidence that the 5′ noncoding region of the chicken lysozyme mRNA is heterogeneous in length. The 5′ termini of the different mRNAs map 29, 31 and 53 nucleotides upstream from their common initiation codon. The 5′ ends of lysozyme-specific nuclear RNAs map at positions similar to that of the mRNA. AT-rich regions and sequences similar to the E. coli RNA polymerase recognition sequence are found around 30 and 70 nucleotides upstream from each of these 5′ termini. The AT-rich regions differ, however, from the canonical Goldberg-Hogness box in that they do not contain the extremely conserved TATA sequence motif. Sequence comparison at the 5′ end of the lysozyme, conalbumin and ovalbumin genes reveals only one region of partial homology, 140 nucleotides upstream from the mRNA start sites.