The anti-sequence, a portable element extending from +1 to +15 of the transcript, is sufficient to prevent promoter escape from a variety of strong final sigma70 promoters. We show here that this sequence does not function with even the strongest final sigma32 promoter. Moreover, a particular class of substitutions in final sigma70 that disrupt interaction between Region 2.2 of final sigma70 and a coiled-coiled motif in the beta'-subunit of RNA polymerase antagonizes the function of the anti-element. This same group of mutants prevents lambdaQ-mediated anti-termination at the lambdaP(R') promoter. At this promoter, interaction of final sigma70 with the non-template strand of the initial transcribed sequence (ITS) is required to promote the pause prerequisite for anti-termination. These mutants prevent pausing because they are defective in this recognition event. By analogy, we suggest that interaction of final sigma70 with the non-template strand of the anti-ITS is required for function of this portable element, thus explaining why neither final sigma32 nor the Region 2.2 final sigma70 mutants mediate anti-function. Support for the analogy with the lambdaP(R') promoter comes from preliminary experiments suggesting that the anti-ITS, like the lambdaP(R') ITS, is bipartite.

Transcription is delayed in the leader regions of the Escherichia coli trp and his operons by multipartite pause signals that consist of four components: a nascent RNA structure (the pause hairpin), the 10 or 11 nt 3′-proximal region between the pause hairpin and the RNA 3′ end, the bases in the active site, and ∼14 bp of duplex DNA downstream from the pause site. Results described in the accompanying paper suggest that the his pause hairpin slows nucleotide addition via interaction with an easily disordered surface on RNA polymerase. Here we report that the four pause signal components slow nucleotide addition in a single kinetic intermediate. Formation of the paused transcription complex, in contrast, involves synergistic effects of RNA and DNA sequences that select the wild-type pause site from among several adjacent possibilities. Extending the pause hairpin with one G·C base-pair reduces pausing, apparently by interfering with pause hairpin interaction; adding a second C·G base-pair that reduces the 3′-proximal RNA to 9 nt or less (within the 7 to 9 nt characteristic of ρ-independent terminators) induces transcript release. We propose that escape from the pause is governed by a rate-limiting isomerization that may require substrate NTP binding to re-establish the active site geometry, whereas transcript release and termination ensue when the hairpin interaction is weakened and isomerization to an active conformation is blocked.

We examined the effects of neutral salts and the non-ionic solute 2-methyl,-2,4-pentanediol (MPD) on transcript elongation by Escherichia coli RNA polymerase and on pausing induced by the multipartite his leader pause signal. All solutes tested slowed the overall rate of elongation, with anions showing the dominant effects in the order: (most inhibitory) HPO4(2-) > OAc- > SO4(2-) > ClO4- > I- approximately NO3- > Br approximately Cl- approximately MPD (least inhibitory). Although the protein structure-stabilizing anions HPO4(2-), OAc-, and SO4(2-) also increased the pause half-life at the his leader pause site, the remaining solutes accelerated escape from pause site in the order: (greatest acceleration) NO3- > ClO4- > I- > Br- > Cl- > MPD (least acceleration). Cl(-)-induced acceleration of escape from the pause site also occurred on mutant templates altered for the 3'-proximal region, RNA 3' end, or downstream DNA. The effect was eliminated, however, by base substitutions that destabilize the pause RNA hairpin or that extend it toward the 3' end. This "perfect hairpin" itself reduced the pause half-life by a factor of 3. We suggest that the pause RNA hairpin stabilizes a paused conformation of the transcription complex through an interaction with an easily disordered region of RNA polymerase. Extending the stem of the pause hairpin may disrupt the interaction by altering the position of the hairpin in the transcription complex. Anions may either compete for the interaction directly or disorder the site of hairpin interaction by chaotropic effects. We suggest that the negative effect of structure-stabilizing anions like OAc- and SO4(2-) may reflect passage of RNA polymerase through significantly different conformations during rapid elongation, some of which may expose hydrophobic surface.

This chapter discusses the quantitative analysis of transcriptional pausing by Escherichia coli (E. coli) RNA polymerase. DNA-dependent RNA polymerases do not synthesize RNA chains at a constant rate. Instead, rapid elongation through some segments of DNA is punctuated by pausing at others. At pause sites, RNA polymerase adds the next NTP at least 100 times more slowly than its optimal elongation rate—that is, ∼75 nucleotides per second both in vivo when pausing is suppressed and in vitro on the nonpausing template poly[d(AT)]. Some pause sites occur at strategic positions in transcriptional units where they halt elongation and facilitate the interaction of RNA polymerase or the nascent RNA with regulatory factors. These factors may trigger termination directly (e.g., Rho), regulate a subsequent termination event, alter the pathway of RNA folding or release RNA polymerase from the paused state. The chapter describes basic methods to measure pausing, using E. coli his-leader pause site as a paradigm. These methods can be used to test the mechanism by which RNA polymerase recognizes and escapes from pause sites, and to assess the effects of transacting factors on it. Both the experimental approach and the methods of data analysis are applicable to eukaryotic RNA polymerases as well, for which pausing also appears to play regulatory roles. This his-leader pause site stops transcription midway through an operon leader region that includes a leader-peptide-coding region and a termination site (the attenuator). Pausing delays the arrival of RNA polymerase at the attenuation decision point until a ribosome can release it and control the attenuation decision.

The recent publication of the 2.6 å crystal structure of a portion of σ70 provides insight into the role of sigma during transcription initiation. This high resolution picture unveils novel questions.

A central enigma of transcriptional regulation is how the normally efficient transcription elongation complex stops at pause and termination signals. One possibility, raised by the discovery that RNA polymerase sometimes contracts its DNA footprint, is that discontinuous movements contribute to recognizing these signals. We report that E. coli RNA polymerase responds to sequences immediately downstream and upstream from the his leader pause site by changing neither its downstream DNA contact nor its upstream RNA contact for 8 bp preceding the pause. This compressed complex isomerizes to a paused conformation by an approximately 10 bp jump of its downstream DNA contact and simultaneous extrusion of an RNA hairpin that stabilizes the paused conformation. We suggest pausing and termination could be alternative outcomes of a similar isomerization that depend on the strength of contacts to 3'-proximal RNA remaining after the jump.

The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.

A key feature of transcriptional attenuation in some amino acid biosynthetic operons is a transcriptional pause that occurs immediately after synthesis of the first leader transcript secondary structure. Both RNA secondary structure and downstream DNA sequence are important for pausing at these sites; however, the precise RNA structures involved and the relative contribution of other RNA and DNA bases to pausing are unknown. We studied the effects of base substitutions upstream from the his leader pause site (immediately prior to addition of G103) to determine how nucleic acid sequences and RNA structure contribute to pausing. By testing compensatory base substitutions, we found that pausing depended in part on an RNA secondary structure containing a five base-pair stem and eight nucleotide loop, which we call the his pause RNA hairpin. The his pause hairpin forms 11 nucleotides upstream from the paused transcript 3′ end and thus corresponds to only the upper portion of the larger his A : B leader transcript secondary structure. Some base substitutions in the ten nucleotides between the pause hairpin and the 3′ end of the transcript increased pausing, whereas others decreased pausing. However, compensatory substitutions that restored pairing of these bases in the lower portion of the A : B secondary structure did not alter these effects. Changing the 3′-terminal nucleotide of the transcript (U102) altered both the position and strength of pausing. Thus, in addition to the downstream DNA sequence, three distinct segments of nucleic acid upstream from the nucleotide-addition site in the transcription complex contribute to pausing in different ways: the pause RNA hairpin, the 3′-proximal region of transcript or DNA template, and the 3′-terminal nucleotide. We suggest that electrostatic interaction between the pause hairpin and RNA polymerase, rather than disruption of an RNA : DNA heteroduplex, delays elongation at the his leader pause site.

The Salmonella typhimurium his leader region contains a well documented transcription attenuator. We report here the results of in vitro transcription studies that characterized a transcription pause site in the his leader region. The pause occurred after synthesis of the first his leader secondary structure (A:B) and immediately preceding addition of G103 to the nascent transcript. RNA polymerase pausing at this site would allow a ribosome synthesizing the his leader peptide to release the paused polymerase and synchronize transcription and translation of the his leader region. The half-life of transcription complexes paused in the his leader was enhanced by NusA, but not guanosine 5'-diphosphate 3'-diphosphate. Nuclease digestion and RNA modeling studies were consistent with a compact three-dimensional structure for the his pause RNA. The half-life of the his leader paused transcription complex was decreased greatly on altered templates in which the C71-G93 base pair was disrupted but was unchanged when the C65-G100 base pair was disrupted. This result is consistent with a model for the structure of paused transcription complexes in which a portion of the RNA:DNA elongation heteroduplex is retained.