Nus A is involved in transcriptional termination on lambda tI.

The transcriptional terminator tI generates the 3'end of the integrase (int) gene transcript that is read from the lambda PI promoter in lambda phage. We have studied the factors that affect transcription termination in vitro and in vivo at the lambda tI terminator. In vitro transcriptional studies showed that tI is about 80% efficient in the presence of purified NusA protein, whereas it is only about 50% efficient in its absence. In vivo studies, where the readthrough transcript of lambda tI was measured by quantitative dot blot analysis, gave about 80% efficiency in wild-type strains, but only 60% in the nusA1 mutant strain at non-permissive temperatures. These results support the idea that termination at lambda tI in vivo involves interaction with the NusA factor.

Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes.

Ternary complexes of RNA polymerase containing the DNA template and nascent RNA are the intermediates in transcript elongation in all cells. We have footprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase. When complexes are treated with elevated levels of ribonucleases A and T1, the nascent transcript can be cleaved to within 3-4 nucleotides of the 3'-terminus. Ternary complexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an active ternary complex. However, cleavage by ribonuclease I is restricted, and gives a limited digest product of about 16 nt. At lower concentrations of ribonuclease T1, two regions of partial protection are seen. The first region extends through the first 15-16 nucleotides from the 3'-OH terminus; the second region extends from position 30 out to position 45. We interpret these regions of partial protection as defining two RNA product binding sites on the RNA polymerase that bind the product to the enzyme during elongation. Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length.

Basic mechanisms of transcript elongation and its regulation.

Ternary complexes of DNA-dependent RNA polymerase with its DNA template and nascent transcript are central intermediates in transcription. In recent years, several unusual biochemical reactions have been discovered that affect the progression of RNA polymerase in ternary complexes through various transcription units. These reactions can be signaled intrinsically, by nucleic acid sequences and the RNA polymerase, or extrinsically, by protein or other regulatory factors. These factors can affect any of these processes, including promoter proximal and promoter distal pausing in both prokaryotes and eukaryotes, and therefore play a central role in regulation of gene expression. In eukaryotic systems, at least two of these factors appear to be related to cellular transformation and human cancers. New models for the structure of ternary complexes, and for the mechanism by which they move along DNA, provide plausible explanations for novel biochemical reactions that have been observed. These models predict that RNA polymerase moves along DNA without the constant possibility of dissociation and consequent termination. A further prediction of these models is that the polymerase can move in a discontinuous or inchworm-like manner. Many direct predictions of these models have been confirmed. However, one feature of RNA chain elongation not predicted by the model is that the DNA sequence can determine whether the enzyme moves discontinuously or monotonically. In at least two cases, the encounter between the RNA polymerase and a DNA block to elongation appears to specifically induce a discontinuous mode of synthesis. These findings provide important new insights into the RNA chain elongation process and offer the prospect of understanding many significant biological regulatory systems at the molecular level.

Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates.

Several models have been proposed for the mechanism of transcript termination by Escherichia coli RNA polymerase at rho-independent terminators. Yager and von Hippel (Yager, T. D. & von Hippel, P. H. (1991) Biochemistry 30, 1097-118) postulated that the transcription complex is stabilized by enzyme-nucleic acid interactions and the favorable free energy of a 12-bp RNA-DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble. Termination, by their model, is viewed simply as displacement of the RNA transcript from the hybrid helix by reformation of the DNA helix. We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interactions with two single-strand specific RNA-binding sites; termination is triggered by formation of an RNA hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately, leads to its ejection from the complex. To distinguish between these models, we have tested whether E. coli RNA polymerase can terminate transcription at rho-independent terminators on single-stranded DNA. RNA polymerase cannot form a transcription bubble on these templates; thus, the Yager-von Hippel model predicts that intrinsic termination will not occur. We find that transcript elongation on single-stranded DNA templates is hindered somewhat by DNA secondary structure. However, E. coli RNA polymerase efficiently terminates and releases transcripts at several rho-independent terminators on such templates at the same positions as termination occurs on duplex DNAs. Therefore, neither the nontranscribed DNA strand nor the transcription bubble is essential for rho-independent termination by E. coli RNA polymerase.

Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus.

We have studied a novel class of DNA sequences that cause DNA polymerases to pause. These sequences have the central consensus Py-G-C and are not necessarily adjacent to hairpins in the DNA template. Since most consensus sequences do not cause pauses under standard conditions, additional template features must exist that make it difficult to incorporate nucleotides at these positions. We believe that these pauses result from constraints that make the conformation change involved in nucleotide selection more difficult. These pauses can obscure parts of DNA sequencing ladders and prevent DNA amplification by the polymerase chain reaction. The addition of betaine, and some related compounds, relieves these pauses.

Escherichia coli fliAZY operon.

We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY. Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro. We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters. FliZ and FliY are not required for motility but may regulate sigma F activity, perhaps in response to a putative cell density signal that may be detected by FliY, a member of the bacterial extracellular solute-binding protein family 3.

Escherichia coli transcript cleavage factors GreA and GreB stimulate promoter escape and gene expression in vivo and in vitro.

The process of RNA chain initiation by RNA polymerases plays a central role in the regulation of transcription. In this complex phase of transcription, short oligomers are synthesized and released from the enzyme-promoter complex in a reaction termed abortive initiation. The polymerase undergoes many cycles of abortive initiation prior to completion of the initiation process, which is signaled by the translocation of the enzyme away from the promoter, release of sigma factor, and formation of an elongation complex in which the RNA is stably bound. We have studied the parameters that affect escape from the promoter by Escherichia coli RNA polymerase for the phage T7 A1 promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25antiDSR. The latter site contains a synthetic initial transcribed region that reduces its ability to synthesize RNA both in vivo and in vitro. Clearance from T5 N25antiDSR can be stimulated up to 10-fold in vitro by addition of the E. coli transcript cleavage factor GreA or GreB, but these factors have little effect on transcription from the normal T7 A1 or T5 N25 promoters. Using an E. coli strain lacking GreA and GreB, we were also able to show stimulation of transcription by the Gre factors from the T5 N25antiDSR promotor in vivo. The stimulation of RNA chain initiation by Gre factors, together with their known biochemical properties in the transcription elongation reaction, suggests some specific models for steps in the transcription initiation reaction.

Identification of flagellar synthesis regulatory and structural genes in a sigma D-dependent operon of Bacillus subtilis.

The sigma D form of RNA polymerase from Bacillus subtilis has been shown previously to direct the synthesis of several transcription units bearing genes for flagellin, motility proteins, and autolysins. In this report, we describe an operon of genes transcribed from the sigma D-dependent promoter PD-1. We have identified three complete open reading frames and one partial one downstream of this promoter; immediately upstream is the previously identified comF locus. The PD-1 operon encodes the presumptive B. subtilis homologs of two Salmonella typhimurium late flagellar genes, flgM and flgK. Also present in this operon are two genes of unknown function, orf139 and orf160, whose products show similarities to the eukaryotic cytoskeletal proteins myosin and vimentin, respectively. orf139 and orf160 may encode proteins that form extended alpha-helical secondary structures and coiled-coil quaternary structures which may be filamentous components of the gram-positive bacterial flagellum. We have characterized the B. subtilis flgM gene further by constructing an in-frame deletion mutation, flgM delta 80, and creating strains of B. subtilis in which this allele has replaced the wild-type copy. By primer extension analysis of cellular RNA, we have shown that the flgM delta 80 mutation relieves the block to transcription of two other sigma D-dependent operons imposed by an unlinked mutation in a gene directing early flagellar synthesis. We conclude that, as in the case of S. typhimurium, early flagellar synthesis in B. subtilis is coupled to late flagellar synthesis through repression of sigma D-dependent transcription by the flgM gene product.

Characterization of the sigD transcription unit of Bacillus subtilis.

The sigma D factor of Bacillus subtilis is required for the transcription of the flagellin and motility genes as well as for wild-type chemotaxis. Southern blot and sequence analyses demonstrate that the structural gene for sigma D, sigD, is located immediately downstream of a region of DNA originally identified as the chemotaxis (che) locus and now renamed the fla/che region. In fact, sigD appears to be part of a very large operon (> 26 kb) containing genes which encode structural proteins that form the hook-basal body complex as well as regulatory proteins required for chemotaxis. Transposon insertions up to 24 kb upstream of sigD, within several of the genes for the hook-basal body components, give rise to only a moderate decrease in sigD expression. The transposon insertions, however, block sigma D activity as demonstrated by the lack of flagellin expression in strains bearing these insertions. These effects appear to arise from two types of regulation. In cis the transposon insertions appear to introduce a partial block to transcription of sigD from upstream promoter elements; in trans they disrupt genes whose gene products are required for sigma D activity. It appears that sigD transcription is initiated, at least in part, by a promoter many kilobases upstream of its translation start site and that transcription of the flagellin gene by sigma D is dependent on the formation of a functional hook-basal body complex. The possibility that sigD is part of the fla/che operon was further tested by the integration of an insertion plasmid, containing strong transcription terminators, 1.6 and 24 kb upstream of the sigD gene. In both cases, the introduction of the terminators resulted in a greater decrease of sigD expression than was caused by the plasmid sequences alone. These results indicate that wild-type transcription of sigD is dependent on promoter sequences > 24kb upstream of its structural gene and that the entire fla/che region forms a single operon.

Regulation of sigma D expression and activity by spo0, abrB, and sin gene products in Bacillus subtilis.

Expression of sigma D protein and of the hag gene, which is transcribed by the sigma D holoenzyme, is not dependent on spo0, abrB, or sin gene products in Bacillus subtilis. Preliminary results, however, suggest that a signal mediated by the spo0K locus may be responsible for the inhibition of sigma D activity during the stationary phase.

Complexes of yeast RNA polymerase II and RNA are substrates for TFIIS-induced RNA cleavage.

In the absence of DNA, purified yeast RNA polymerase II can bind RNA to form a binary complex. RNA in such RNA-RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA, including TFIIS-dependent cleavage and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by alpha-amanitin. Hence, by several criteria the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes in which the catalytic site for nucleotide addition is positioned at or near the 3'-OH terminus of the RNA. These findings are consistent with a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein-binding sites located up to 10 nt apart.

RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein.

Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex.

Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.

An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase.

Two genes controlling motility functions in Bacillus subtilis were identified by DNA sequence analysis of a chromosomal fragment containing a strong promoter for sigma D RNA polymerase. Previous studies had shown that this sigma D-dependent promoter controls synthesis of a 1.6-kb transcript in vivo and in vitro. Sequence analysis revealed that the 1.6-kb transcript contains two open reading frames coding for protein sequences homologous to the Escherichia coli motA and motB gene products, respectively, and ends in a rho-independent termination site. Direct evidence linking these genes to motility functions in B. subtilis was obtained by precise localization by polymerase chain reaction of Tn917 transposon insertion mutations of Mot- strains, isolated by Zuberi et al. (A. R. Zuberi, C. Ying, H. M. Parker, and G. W. Ordal, J. Bacteriol. 172:6841-6848, 1990), to within this mot. operon. Replacement of each wild-type gene by in-frame deletion mutations yielded strains possessing paralyzed flagella and confirmed that both motA and motB are required for the motility of B. subtilis. These current findings support our earlier suggestions that sigma D in B. subtilis plays a central role in the control of gene expression for flagellar assembly, chemotaxis, and motility functions. Sigma F, the enteric homolog of sigma D, controls similar functions in E. coli and Salmonella typhimurium, and these factors appear to be representative of a family of factors implicated in flagellar synthesis in many bacterial species, which we propose to designate the sigma 28 family.

Sequence and characterization of Bacillus subtilis CheW.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.

Structural analysis of ternary complexes of Escherichia coli RNA polymerase. Individual complexes halted along different transcription units have distinct and unexpected biochemical properties.

Ternary complexes containing RNA polymerase, DNA and nascent RNA are intermediates in all RNA syntheses and are the targets of cellular factors that regulate RNA chain elongation and termination. Hence, elucidation of the structure and properties of these complexes is essential for understanding the catalytic and regulatory properties of the enzyme. We have described methods to prepare ternary complexes halted at defined positions along the DNA template, using specific dinucleotides to prime chain initiation along with limited subsets of the NTP substrates. Study of these static, halted complexes may provide information about the structure and properties of the transient elongation intermediates involved in transcription, although there is no necessary direct relationship between the two. Using specific halted complexes as precursors, we have walked the RNA polymerase along its template, producing defined ternary complexes at unique sites along two different transcription units. These complexes differ significantly from one another in many biochemical properties, in dramatic contrast to the properties expected from models that postulate a monotonous structure for elongation intermediates. These differences include variations in complex mobility during electrophoresis in non-denaturing polyacrylamide gels, in thermal stability and in stability to dissociation. Some halted complexes lose the ability to resume elongation when presented with the missing substrates. These "dead end" complexes must represent metastable structures in which elongation is blocked, and demonstrate clearly that not all halted complexes can be considered true intermediates in elongation. Other halted complexes rapidly cleave the nascent RNA seven nucleotides from the 3' terminus, in an unexpected and unusual biochemical reaction. These differences in properties among complexes bearing transcripts that differ by only one or a few nucleotides suggest that they have distinct structures. These differences must be due, at least in part, to differences in the template sequence and the length of the transcript. The results raise important questions as to the actual mechanism of transcription elongation, and suggest that it is a much more complex process than previously assumed.

Structural analysis of ternary complexes of Escherichia coli RNA polymerase. Deoxyribonuclease I footprinting of defined complexes.

The structure and properties of ternary complexes of RNA polymerase are of central importance in understanding the mechanisms of transcriptional elongation and termination, and the regulation of these primary steps in gene expression. However, there has been no systematic study of the structure and properties of such complexes along a single transcription unit. Recently, we have described the isolation of a collection of halted ternary complexes of Escherichia coli RNA polymerase bearing transcripts from 11 to 35 nucleotides in length along two different transcription units (accompanying paper). Here, we report structural studies of these complexes using DNase I footprinting. Surprisingly, nearly all of the different ternary complexes have distinctly different footprints along the two DNA strands, and the position of the footprint relative to the 3' end of the transcript also varies for most complexes. Halted complexes bearing transcripts of comparable size do not have identical footprints; hence, DNA sequence as well as transcript length plays a role in determining the size and position of the footprint. These differences in structure are consistent with our earlier findings that ternary complexes can differ considerably in stability and gel mobility. The downstream boundary of the RNA polymerase in ternary complexes does not move forward regularly as successive nucleotide residues are added to the RNA chain. In contrast, the upstream boundary moves forward more or less in concert with the movement of the 3' terminus of the transcript. These factors lead to a general compression of the overall footprint as transcription proceeds, together with a steady movement of the 3' terminus of the RNA toward the downstream boundary of the polymerase. Ultimately, after the length of the RNA transcript has increased from eight to ten nucleotides, the downstream boundary of the complex is found to move downstream along the DNA, suggesting a translocation event. We suggest that RNA chain elongation, like RNA chain initiation, may involve a saltatory process in which net translocation of the complex along the DNA occurs only after addition of a number of ribonucleotides to the RNA chain.

Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators.

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Parameters affecting transcription termination by Escherichia coli RNA. II. Construction and analysis of hybrid terminators.

Rho-independent terminators are characterized by two major functional regions, one upstream from the termination site having a sequence capable of forming an RNA hairpin in the nascent transcript, the second extending, from the base of this hairpin, seven to nine nucleotides along the transcript to the actual sites of termination (3'-tail region). This latter region of the transcript is often rich in uridine residues. Both regions are postulated to play central roles in the termination process. We have constructed a series of hybrid rho-independent, transcription terminators in which sequences upstream and downstream from the RNA hairpin for the Escherichia coli trp attenuator (trpatt+) are interchanged with sequences from trpatt mutant (1419) or from the phage T7 early terminator (T7Te). Similar hybrids have been constructed for T7Te, replacing flanking sequences with trpatt regions. The effects of such changes on transcription termination have been tested in vitro with purified E. coli RNA polymerase to determine the intrinsic termination efficiency (%T) of each hybrid terminator. Both the trpatt+ terminator and T7Te are highly efficient rho-independent terminators in vitro. However, replacement of trpatt+ sequences upstream and downstream from the RNA-terminator hairpin with the comparable T7Te sequences reduces %T dramatically, suggesting that the RNA-terminator hairpin does not function independently from its flanking regions. Regions downstream from the actual termination/release site are shown to be of considerable importance in determining %T for terminators bearing the T7Te or trpatt1419 3'-tail region, but have little effect on terminators with the trpatt+ 3'-tail region. For terminators bearing the T7Te or trpatt1419 3'-tail region that are inefficient, efficient termination is restored by elevated concentrations of KCl in the reaction. The results do not fit well with models for termination in which %T is determined by a two-step process in which the terminator-RNA hairpin, and a seven to 12 base-pair DNA-RNA hybrid structure rich in uridine residues, act independently to cause the polymerase to pause, and to release the transcript, respectively. DNA sequences both upstream and downstream from these regions, as well as DNA sequences downstream from the transcript termination site, can significantly affect the termination process. Conversely, terminators lacking a 3'-tail region rich in uridine residues can be highly efficient, but only when joined with appropriate sequence immediately downstream from the termination site. This suggests that the 3'-tail region acts in some manner other than the formation of an unstable DNA-RNA hybrid that facilitates termination.