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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.

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We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.

The Bacillus subtilis gene hag, which codes for the flagellin structural protein, was identified by DNA sequence analysis in a collection of DNA fragments bearing in vitro promoters for the c28 form of RNA polymerase. The hag gene and adjacent, regions of the B. subtilis chromosome were restriction mapped, and the nucleotide sequence was determined. The hag gene was transcribed at all stages of growth from a single promoter that had sequences in the promoter recognition region characteristic of the consensus sequence for the o28 holoenzyme. Transcription of hag was elinminated by insertion mutations that blocked synthesis of the I28 protein. These findings provide strong support for the previous proposal that the Cr28 form of RNA polymerase controls transcription of a regulon specifying flagellar, chemotaxis, and motility functions in B. subtilis (J. D. Helmann and M. J. Chamberlin, Proc. Natl. Acad. Sci. USA 84:6422-6424, 1987). The steady-state levels of hag mRNA increased during exponential growth and peaked as the B. subtilis cells entered the stationary phase. The transcript levels then decreased to zero within 4 h after the onset of sporulation. Hence, cr28 RNA polymerase function is temporally regulated.

Bacillus subtilis contains multiple forms of RNA pol,ymerase holoenzyme, distinguished by the presence of different specificiy determinants known as (r factors.. The au factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the a8 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the a8 -structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the a8 gene was determined, and the cloned 28 (F gene was used to construct a mutant strain which does not express the ('8 protein. This strain also failed to synthesize flageilin protein and grew as long filaments. The predicted (28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The d8 protein sequence was similar to that of other sequenced 'r factors and to theflbB gene product of Escherichia coli. Since theflbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that cr8 functions to regulate flagellar synthesis in B. subtilis.Bacterial RNA polymerase is a multisubunit enzyme central to the process of gene expression. Although the catalytic activity resides in the core subunits of the enzyme, the promoter specificity of a particular holoenzyme is determined by the nature of the associated a factor (31). The majority of cellular transcription is dependent on the primary r factor, which exhibits a conserved promoter recognition specificity throughout the eubacteria (43). Many bacterial species also contain alternative a factors that are specific for transcription of distinct regulons of coordinately regulated genes. These alternative a factors normally recognize promoter sequences that are differett from those recognized by the primary cr factor. Examhples of alternative of factors in the enteric bacteria include a32 (15), specific for the transcription of heat shock genes, and (94 (19, 20), specific for transcription of fnitrogen-regulated genes. For Bacillus subtilis, at least six alternative factors have been described (26,31).In B. subtilis, alterations in cellular transcription, mediated at least in part by alternative cr factors, effect the precise temporal changes in gene expression necessary for endospore formation (26). The products of the spoOH (U30) and spoIIGB (r29) genes are sporulation-specific cr factors that have been characterized both genetically and biochemically (6,22,24,26,40,41). In addition, the spoIIAC gene product is homologous to other sequenced a factors (9) and may also function as a cf factor. The cr3 and a98 factors are found in vegetatively growing cells and are dispensable for sporulation, since disruption of these genes does not impair sporulation (3, 8; see below). To define the biological function of the B. subtilis ur28 factor, we have begun a genetic and structural analysis of the Cr28 structural gene (sigD) bas...