PubMed Health⌕ Search

Biomedical subjects

J D Gralla

Publications and source records attributed to J D Gralla.

At least 19 recordsLinked to original sources
PubMed2001

Function of the bacterial TATAAT -10 element as single-stranded DNA during RNA polymerase isomerization.

The bacterial TATAAT -10 region sequence was the first promoter element to be identified, but how it functions is still not clear. Because the duplex element is melted during initiation, the effects of substitutions were studied in both single-and double-strand contexts. Band-shift results were particularly unexpected in the context of melted DNA. The effect of the lac UV5-melted -10 region on polymerase binding was found to include a large sequence nonspecific contribution. Instead the dominant role of single-stranded -10 region nucleotides was in directing the isomerization of the RNA polymerase to its heparin resistant form. This role becomes minimal when the melting is extended beyond the -10 region to encompass the transcription start site, as in the final open complex. The duplex binding results are in agreement with previous reports that showed positions -12T and -11A are of primary importance for promoter recognition. Thus the consensus -10 region sequences function in two ways, both before full promoter melting. They stabilize initial polymerase binding via duplex interactions and subsequently as single-stranded DNA they promote enzyme isomerization to the functional form.

Base Sequence↗
PubMed2001

Sigma38 (rpoS) RNA polymerase promoter engagement via -10 region nucleotides.

Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (-12T and -11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the -10 region (CTAcacT from -13 to -7). The remaining two nucleotides were shown to have different roles, -13C in preventing recognition by the heterologous sigma70 polymerase and -7T in directing enzyme isomerization. These -10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.

Bacterial Proteins↗
PubMed2000

Roles for the C-terminal region of sigma 54 in transcriptional silencing and DNA binding.

Twenty-one conserved positively charged and aromatic amino acids between residues 331 and 462 of sigma 54 were changed to alanine, and the mutant proteins were studied by transcription, band shift analysis, and footprinting in vitro. A small segment corresponding to the rpoN box was found to be most important for binding duplex DNA. Two amino acids, 52 residues apart, were found to be critical for maintaining transcriptional silencing in the absence of activator. These two activator bypass mutants and several other mutants failed to bind the type of fork junction DNA thought to be required to maintain silencing. The two bypass mutants showed a binding pattern to DNA probes that was unique, both in comparison to other C-terminal mutants and to previously known N-terminal bypass mutants. On this basis, a model is proposed for the role of the C terminus and the N terminus of sigma 54 in enhancer-dependent transcription.

DNA↗
PubMed2000

TATA-flanking sequences influence the rate and stability of TATA-binding protein and TFIIB binding.

The kinetics of TATA-binding protein (TBP) and TFIIB binding were measured on a series of promoter constructs that had varying sequences within and flanking the TATA box. The flanking sequences were found to influence TBP stability even though they do not contact the protein. This occurs by altering the decay rate rather than the association rate. TFIIB association is accompanied by protein-protein cooperativity as indicated by the simultaneous release of both proteins in challenge experiments. The sequence of the TATA box and the sequences that flank it can influence the kinetics of the TFIIB.TBP.DNA complex. TFIIB can contribute to tighter TATA binding in two ways. It always slows the decay rate of TBP, but it can also increase the rate of association at promoters with certain combinations of TATA and flanking sequences. The results imply that the interplay between the TATA box and flanking elements leads to variations in the kinetics of preinitiation complex formation that may account for the observed effects of all of these diverse sequences on transcription.

DNA↗
PubMed2000

Promoter opening by sigma(54) and sigma(70) RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control.

Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that sigma(54) imposes tighter regulation by creating a complex molecular switch at -12/-11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes sigma(54) to alter its cross-linking pattern near -11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent sigma(70) promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two sigma factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.

Adenine↗
PubMed2000

Escherichia coli promoter opening and -10 recognition: mutational analysis of sigma70.

The opening of specific segments of DNA is required for most types of genetic readout, including sigma70-dependent transcription. To learn how this occurs, a series of single point mutations were introduced into sigma70 region 2. These were assayed for duplex DNA binding, DNA opening and DNA double strand-single strand fork junction binding. Band shift assays for closed complex formation implicated a series of arginine and aromatic residues within a minimal 26 amino acid region. Permanganate assays implicated two additional aromatic residues in DNA opening, known to form a parallel stack of the type that can accept a flipped-out base. Substitution for either of these aromatics had no effect on duplex probe recognition. However, when a single unpaired -11 nucleotide is added to the probe, the mutants fail to bind appropriately to give heparin resistance. A model for DNA opening is presented in which duplex recognition by regions 2.3, 2.4 and 2.5 of sigma positions the pair of aromatic amino acids, which then create the fork junction required for stable opening.

Bacterial Proteins↗
PubMed2000

Roles for non-TATA core promoter sequences in transcription and factor binding.

Sequence blocks within the core region were swapped among RNA polymerase II promoters to explore effects on transcription in vitro. The pair of blocks flanking TATA strongly influenced general transcription, with an additional effect on promoter activation. These flanking elements induced a change in the ratio of activated to basal transcription, whereas swapping TATA and initiator sequences only altered general transcription levels. Swapping the flanking blocks influenced binding by general transcription factors TBP and TFIIB. The results suggest that the architecture of the extended core sequence is important in determining promoter-specific effects on both general transcription levels and the tightness of regulation.

Adenovirus E4 Proteins↗
PubMed1999

The use of ATP and initiating nucleotides during postrecruitment steps at the activated adenovirus E4 promoter.

Permanganate probing has been used to follow the progress and ATP dependence of promoter opening during activated adenovirus E4 initiation and clearance. Using templates designed to restrict synthesis to defined positions, formation of a 3-nucleotide-long RNA was found to be sufficient to trigger expansion of the initial transcription bubble. This occurred by a discrete transition that expanded the downstream limit of melting from position 1 to 15. Subsequent clearance of the bubble from the promoter region also occurred without detectable intermediates. Thus, initial opening, extension, and the clearance of the promoter bubble appear to occur as discrete, unique transitions. The apparent K(m) values for these three steps were determined to be near 5, 9, and 50 microM, respectively. Comparison of these values with ATPase activities within known transcription factors raises the possibility that different activities could be responsible for each step.

Adenosine Triphosphate↗
PubMed1999

A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor.

Results of binding assays using DNA fork junction probes indicate that sigma 54 contains multiple determinants that regulate melting to allow RNA polymerase to remain in closed promoter complexes in order to respond to enhancers. Gel mobility shift studies indicate that the -12 promoter element and parts of sigma 54 act together to form a molecular switch that controls melting. The DNA sequences and the sigma 54 N-terminus help direct polymerase to the location within the -12 promoter element where melting will initiate. However, the fork junction that would lead to melting does not form, due to the action of an inhibitory DNA element. Such unregulated melting is inhibited further by the lack of availability of the single-strand binding elements, which are needed to spread opening from the junction to the transcription start site. Thus, in the absence of looping enhancer protein, proper regulation is maintained as the sigma 54 polymerase remains bound in an inactive state. These complex protein-DNA interactions allow the controls over protein recruitment and DNA melting to be separated, enhancing the diversity of accessible mechanisms of transcription regulation.

Base Pairing↗
PubMed1999

Transcription reinitiation rate: a potential role for TATA box stabilization of the TFIID:TFIIA:DNA complex.

Potential pathways that could account for observed rapid rates of transcription reinitiation were explored. A nuclear extract system was established in which reinitiation rates were observed to be kinetically facilitated and in which the rate was sensitive to TATA box mutation. Kinetic facilitation of functional complex formation could be mimicked by pre-assembling activator and certain general transcription factors on the promoter and then adding nuclear extract. The minimal activated complex with this characteristic contained general factors TFIID and TFIIA. The ability of the TFIID:TFIIA complex to complete assembly rapidly was reduced by the same TATA box mutation that reduced reinitiation rate. Band shift experiments also showed that this same mutation lowered the stability of the TBP:TFIIA complex on the DNA. The results suggest that TATA-dependent variations in retention of the TFIID:TFIIA complex after release of the polymerase could be a primary determinant of reinitiation rate, allowing diversity in promoter strength to be related to diversity in TATA element sequences.

DNA↗
PubMed1999

Regulation of sigma 54-dependent transcription by core promoter sequences: role of -12 region nucleotides.

The tetranucleotide core recognition sequence (TTGC) of the sigma 54 promoter -12 recognition element was altered by random substitution. The resulting promoter mutants were characterized in vivo and in vitro. Deregulated promoters were identified, implying that this core element can mediate the response to enhancer-binding proteins. These promoters had in common a substitution at position -12 (consensus C), indicating its importance for keeping basal transcription in check. In another screen, nonfunctional promoters were identified. Their analysis indicated that positions -13 (consensus G) and -15 (consensus T) are important to maintain minimal promoter function. In vitro studies showed that the -13 and -15 positions contribute to closed-complex formation, whereas the -12 position has a stronger effect on recognition of the fork junction intermediate created during open-complex formation. Overall the data indicate that the -12 region core contains specific subsequences that direct the diverse RNA polymerase interactions required both to produce RNA and to restrict this RNA synthesis in the absence of activation.

Consensus Sequence↗
PubMed1998

Promoter opening via a DNA fork junction binding activity.

The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Opening is tightly regulated, but how it occurs is not known. These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening. This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded/single-stranded boundary of the open complex. Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity. The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands. The junction binding activity is contained within the sigma factor component of the holoenzyme. The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP. Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.

Base Sequence↗
PubMed1998

Genomic position analyses and the transcription machinery.

Position analyses have been devised to extract additional transcriptional information from rapidly expanding genomic data bases. The locations of promoter regulatory sites and also the locations of transcription factor DNA-binding domains are analyzed. Strongly preferred positions of activator binding sites occur in both Escherichia coli and eukaryotes, suggesting specific common features of transcription in the two systems. In both systems, regulatory proteins are found to have their DNA-binding domains near termini and the data suggest an evolutionary analysis that complements a phylogenetic analysis based on sequence alignments. The results indicate that positional information can be an important adjunct to sequence comparisons in analyzing genomic information.

Animals↗
PubMed1998

Identification of an N-terminal region of sigma 54 required for enhancer responsiveness.

Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.

Amino Acid Sequence↗
PubMed1998

Multiple in vivo roles for the -12-region elements of sigma 54 promoters.

Alignment of sigma 54-dependent promoters indicates conservation of two sequence elements. Six nucleotides in the downstream -12 element were mutated individually to each nonconsensus nucleotide. mRNA levels were measured in vivo for each promoter under strongly activating conditions. The results showed that the consensus sequence was not the strongest promoter. Instead, the -12 consensus element consists of two subregions that behave differently when mutated. Single changes in the upstream TTT consensus subregion can lead to increases in transcription, whereas single changes in the downstream GC(A/T) can lead to decreases in transcription. Selected double mutations with changes in both subregions were constructed and studied in vivo. No double mutation increased promoter strength, and some decreased it. Mutant promoters were also assayed under nonactivating conditions in vivo. No mRNA was detected in 23 of the 24 promoters tested. However, one double mutant showed substantial levels of transcript, indicating that the -12 sequence was capable of specifying basal transcription under nonactivating conditions. Overall, the results show that the -12 region has multiple roles in transcription in vivo, including modulating both basal and induced RNA levels.

Consensus Sequence↗
PubMed1997

Promoter activation via a cyclic AMP response element in vitro.

Transcription activation via activating transcription factor cyclic AMP response element binding (ATF/CREB) sites in vitro was explored using transcription and permanganate assay for open complex formation. These sites were used to drive transcription from an adenovirus major late core sequence. Under conditions where activation is strong, 20-50-fold, ATF/CREB is required for preinitiation complexes to reach the open complex stage. Complete opening requires activator, ATP, and initiating nucleotides. In exploration of postinitiation steps, no stimulation of promoter clearance was observed but a modest stimulation of the rate of continuous transcription occurred. High amounts of DNA template, commonly used in in vitro studies, allows some templates to open without activator, but leaves the nucleotide requirements intact. This leads to a drastic lowering of the dependence on ATF/CREB. Taken together, the data indicate that ATF/CREB activates this system primarily by stimulating the formation of functional preinitiation complexes.

Activating Transcription Factors↗