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J D Gralla

Publications and source records attributed to J D Gralla.

At least 37 records · Page 2Linked to original sources
PubMed1997

Multiple ATP-dependent steps in RNA polymerase II promoter melting and initiation.

Permanganate probing and abortive initiation assays were used to investigate the role of ATP in several successive stages of transcription initiation at the activated adeno E4 and mouse DHFR promoters. Removal of ATP at several points along the multi-step pathway blocked further progress towards its completion. Most strikingly, even if the DNA transcription start site is opened using ATP, the subsequent removal of ATP disallows formation of the first phosphodiester bond of the RNA. After ATP-dependent formation of a short RNA, a new transcription complex forms, which is more stable and has a longer open region. Both RNA and ATP appear to play roles in the formation of this complex. The need for ATP throughout this multi-step initiation pathway leads to new and unexpected possibilities for the use of energy and ATPases in transcription initiation.

Adenosine Triphosphatases↗
PubMed1997

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: roles for DNA and protein determinants.

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus -12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP beta-gamma hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Amino Acid Sequence↗
PubMed1997

Isolation and properties of enhancer-bypass mutants of sigma 54.

The N-terminal activation domain of Escherichia coli sigma 54 was randomly mutated to provide a library of changes that might allow the required enhancer function to be bypassed. Five clones harbouring mutant sigma factors were obtained that exhibited this property in that they enhanced growth under nitrogen-limiting conditions in cells lacking NtrC. DNA sequence analysis located all mutations to four leucines in a small region between amino acids 25 and 31. No mutant sigma factors retained the hydrophobic character of the leucine residues. Mutant sigma factors were shown to transcribe in vitro without the need for enhancer binding activator or ATP hydrolysis, confirming the in vivo phenotype. These and other data suggest that a very small set of leucines is critical for keeping polymerase function in check, allowing high responsiveness to physiological induction via enhancer proteins such as NtrC.

Adenosine Triphosphate↗
PubMed1997

DNA-binding determinants of sigma 54 as deduced from libraries of mutations.

PCR mutagenesis was used to obtain libraries of mutations in the region between amino acids 300 and 400 in the DNA-binding domain of Escherichia coli sigma 54. Two hundred changes that did not alter function were identified. These were compared with a somewhat smaller number of changes that did alter function. Several important regions were identified. Single point mutations in two of these, near amino acids 363 and 383, destroyed the ability of sigma to bind DNA, as assayed by band shift analysis. A third segment from amino acids 327 to 347 is also a candidate for contributing to DNA binding. Comparison with data in the literature leads to testable proposals for the complex mode of DNA binding that is associated with sigma 54.

Amino Acid Sequence↗
PubMed1996

The transcription initiation pathway of sigma 54 mutants that bypass the enhancer protein requirement. Implications for the mechanism of activation.

In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant forms of sigma 54 shown previously to bypass certain enhancer requirements in vitro. The holoenzymes containing these sigma mutants produce low levels of open complexes at both the glnAp2 and glnHp2 promoters. The open complexes are unusual in that they are destroyed by heparin. Enhancer protein and ATP convert them into a stable heparin-resistant state. The enhancer response occurs over a similar range of NtrC concentration as occurs with the wild-type holoenzyme, indicating that the activation determinants have been largely preserved within these mutants. One-round transcription assays show that the mutant holoenzymes can be driven to transcribe both promoters without NtrC. The unstable opening induced by these mutations apparently serves as a conduit that can shuttle templates into transcriptionally competent complexes. The results lead to a model in which activation occurs in two steps. First, the enhancer complex overcomes an inhibitory effect of the sigma 54 leucine patch and unlocks the melting activity of the holoenzyme. Second, different sigma 54 determinants are used to drive stabilization of the open complexes, allowing the full transcription potential to be realized.

Adenosine Triphosphate↗
PubMed1996

Activation and repression of E. coli promoters.

There is no organism in which transcription initiation is better understood than Escherichia coli. Recent studies using genetics, biochemistry and structure analysis have revealed how RNA polymerase interactions at promoters are regulated. Prominent examples include the recruitment of polymerase by activators touching its alpha and sigma subunits; which subunit is touched depends on which activator is used and where it binds the DNA. The less-common cases centering on enhancer-dependent transcription use an entirely different mechanism, involving either DNA looping or tracking.

DNA-Directed RNA Polymerases↗
PubMed1996

A three-step pathway of transcription initiation leading to promoter clearance at an activation RNA polymerase II promoter.

The progress of transcription bubbles during inhibition in vitro was followed in order to learn how RNA polymerase II begins transcription at the activated adenovirus E4 promoter. The issues addressed include the multiple roles of ATP, the potential effect of polymerase C-terminal domain phosphorylation, and the ability of polymerase to clear the promoter for reinitiation. The results lead to a three-step model for the transition from closed complex to elongation complex, two steps of which use ATP independently. In the first step, studied previously, ATP is hydrolyzed to open the DNA strands over the start site. In a second step, apparently independent of ATP, transcription bubbles move into the initial transcribed region where RNA synthesis can stall. In the third step, transcripts can be made as polymerase is released from these stalled positions with the assistance of an ATP-dependent process, likely phosphorylation of the polymerase C-terminal domain. After this third step, the promoter becomes cleared, allowing for the reinitiation of transcription.

Adenosine Triphosphate↗
PubMed1995

Abortive initiation and first bond formation at an activated adenovirus E4 promoter.

Abortive initiation at the adenovirus E4 promoter was studied by following the production of RNA formed from the initiating nucleotides UpA and CTP. Formation of a specific short RNA via a reaction with appropriate alpha-amanitin sensitivity required promoter, activator, and ATP. In the absence of any of these, an alpha-amanitin-resistant reaction led to lower levels of a product of unknown origin. The alpha-amanitin-sensitive reaction required open promoter complexes, as assayed directly by permanganate probing. This reaction was not blocked by the inhibition of polymerase C-terminal domain kinase activity or by the lack of DNA supercoiling. Thus, formation of the initial bond of the mRNA appears to require activator and ATP to open the DNA but not phosphorylation of the polymerase C-terminal domain. In addition, the abortive initiation reaction was strongly suppressed when all elongation substrates were present, suggesting that cycling to produce high amounts of abortive product is strongly disfavored during productive initiation at this promoter.

Adenosine Triphosphate↗
PubMed1995

Converting Escherichia coli RNA polymerase into an enhancer-responsive enzyme: role of an NH2-terminal leucine patch in sigma 54.

The protein sigma 54 associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation. Here, mutants of sigma 54 enabled polymerases to transcribe without enhancer protein and adenosine triphosphate. The mutations are in leucines within the NH2-terminal glutamine-rich domain of sigma 54. Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch. The results indicate that sigma 54 acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.

Adenosine Triphosphate↗
PubMed1995

Abortive cycling and the release of polymerase for elongation at the sigma 54-dependent glnAp2 promoter.

Transcription initiation at the sigma 54-dependent glnAp2 promoter was studied to follow the state of polymerase as RNA synthesis begins. Sigma 54 polymerase begins transcription in abortive cycling mode, i.e. after the first bond is made, approximately 75% of the time the short RNA is aborted and synthesis must be restarted. Polymerase is capable of abortive initiation until it reaches a position beyond +3 and before +7, at which stage polymerase is released from its promoter contacts and an elongation complex is formed. INitial elongation is accompanied by two transcription bubbles, one moving with the polymerase and the other remaining at the transcription start site. The sigma 54-associated polymerase shows an earlier and more efficient transition out of abortive initiation mode than prior studies of sigma 70-associated polymerase.

Bacterial Proteins↗
PubMed1995

A novel bacterial transcription cycle involving sigma 54.

sigma 54 is the promoter recognition subunit of the form of bacterial RNA polymerase that transcribes from promoters with enhancer elements. DNase footprinting experiments show that sigma 54 is attached selectively to the template strand, which must be single-stranded for transcription initiation. sigma 54 remains bound at the promoter after core polymerase begins elongation, in contrast to the well-established sigma 70-holoenzyme transcription cycle. Permanganate footprinting experiments show that the bound sigma 54 and the elongating core RNA polymerase downstream of it are each associated with a single-strand DNA region. Template commitment assays show that the promoter-bound sigma 54 must be reconfigured before reinitiation of transcription can occur. This unexpected pathway raises interesting possibilities for transcriptional regulation, especially with regard to control at the level of reinitiation.

Bacterial Proteins↗
PubMed1995

Nucleotide requirements for activated RNA polymerase II open complex formation in vitro.

The role of nucleotides in activated RNA polymerase II transcription was studied. Permanganate footprinting confirmed that there is a strict nucleotide requirement for forming open promoter complexes that cannot be overcome by the addition of a dinucleotide primer corresponding to the start site sequence. However, higher concentrations of other nucleoside triphosphates can substitute for ATP in catalyzing open complex formation. Opening catalyzed by these nucleotides is inhibited by the ATP analogue adenosine 5'-O-(thio-triphosphate), suggesting that they may function through cross-binding to the ATP site. The KM for ATP for opening and the involvement of other nucleotides in opening differs from the characteristics reported for TFIIH helicase and C-terminal domain kinase activities. This raises the possibility that opening does not involve these activities. The results alleviate very significantly the considerable current uncertainty concerning the role of ATP in the mammalian mRNA transcription initiation pathway.

Enzyme Activation↗
PubMed1995

PCR mutagenesis identifies a polymerase-binding sequence of sigma 54 that includes a sigma 70 homology region.

Sigma 54 is a minor bacterial sigma factor that is not a member of the sigma 70 family of proteins but binds the same core RNA polymerase. Previously, we identified a region of sigma 54 that is important for binding core polymerase. In this work, PCR mutagenesis was used to identify specific amino acids important for this binding. The results show that important residues are clustered most closely in a short sequence that was previously speculated to be potentially homologous to a sequence in sigma 70. The mutagenesis also identifies important residues in the flanking hydrophobic-acidic region of sigma 54, which is absent in sigma 70. Overall, the data indicate that sigma 54 binds core polymerase through a sequence homologous to that of sigma 70 but in addition uses unique motifs to modify this interaction.

Amino Acid Sequence↗
PubMed1994

RNA polymerase II phosphorylation: uncoupling from GAL4-VP16 directed open complex formation and transcription in a reconstituted system.

An activated transcription system was constructed using substantially purified liver factors, Hela TFIID and GAL4-VP16. The system was used to study the relationship between RNA polymerase II large subunit phosphorylation and other ATP-dependent processes occurring during activated transcription. When C-terminal domain (CTD) kinase activity was inhibited, activator dependent open promoter complex formation proceeded normally. These open complexes could function to produce RNA in the absence of CTD phosphorylation, although the level of RNA produced was changed somewhat. The results demonstrate that RNA polymerase II CTD phosphorylation is not generally required for the formation of activator-dependent, functional open promoter complexes. Taken together with prior results the experiments suggest that a requirement for CTD phosphorylation may be situation-dependent and thus serve a regulatory function.

Adenosine Triphosphate↗
PubMed1994

Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements.

Three synthetic promoters, PS1, PS2 and PS3, which differ in their core promoter elements, were studied in vivo and in vitro. Whereas an increased homology score correlates with higher rates of RNA polymerase binding, it does not correlate with activity in vivo. Permanganate probing in vivo reveals that PS1, which exhibits the lowest homology score, is rate-limited during the early phase of promoter-RNA polymerase interactions. By contrast, PS2 and PS3, with higher homology scores, are limited at a late step involving an open DNA region spanning from +6 to +12, indicating a stalling of RNA polymerase. These complexes disappear upon treatment of cells with rifampicin and are replaced by open complexes covering the start site. Because initiated complexes are selectively insensitive to rifampicin action, this confirms that RNA polymerase stalled at +6 to +12 has initiated RNA synthesis. Kinetic studies indicate that the enzyme is released slowly from this position and that this slow release appears to be responsible for the low promoter activity. For PS3, which exhibits the highest homology score and which binds RNA polymerase most efficiently, the release of the stalled complex is particularly slow. PS3 is found to be the weakest of the three promoters in vivo. These results support models in which promoter activity can be determined by various rate limiting steps, including those following the formation of open complexes and even the initiation of RNA synthesis.

Base Sequence↗
PubMed1994

Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function.

Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three. The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.

Base Sequence↗
PubMed1994

Analysis of the N-terminal leucine heptad and hexad repeats of sigma 54.

In order to assess the role of leucine repeat motifs within bacterial protein sigma 54, a series of point mutants were introduced into the many leucine residues near the N terminus. Functional assays in vivo showed that the leucine residues that comprise the previously identified heptad repeat motif are selectively important for function. These heptad leucine residues are critical for mRNA production and also for recognition of the -12 promoter element. An internal proline substitution destroys the function of the heptad repeat region, suggesting a possible alpha-helical structure. Mutants with changes in the distal part of this N-terminal region show the interesting property of allowing nearly full levels of open complex formation, while nonetheless reducing the level of mRNA transcripts produced. All of the above-mentioned properties differ from those exhibited by mutating the interdigitated glutamine residues, which were previously found to be closely involved in the DNA melting reaction. The collection of data suggests that the N-terminal region contains overlapping functional motifs, hydrophobic heptad and glutamine-rich, which together appear to constitute the activation domain of sigma 54.

Amino Acid Sequence↗