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As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3' ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3' ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3' to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence., (Copyright © 2018 American Society for Microbiology.)

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity.

The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA-binding domain (RNA-BD) of known structure as well as an ATP-binding domain (ATP-BD) for which a structure has been proposed based on homology modeling. A model is proposed in which Rho first makes an interaction with a nascent RNA on a C-rich, primarily single-stranded rut region of the transcript as that region emerges from the exit site of RNA polymerase. A subsequent step involves a temporary release of one subunit of the hexamer to allow the 3' segment of the nascent transcript to enter the central channel of the Rho ring. Actions of the Rho structure in the channel on the 3' segment that are coupled to ATP hydrolysis pull the RNA from its contacts with the template and RNA polymerase, thus causing termination of its synthesis.

A new algorithm called RNAMotif containing RNA structure and sequence constraints and a thermodynamic scoring system was used to search for intrinsic rho-independent terminators in the Escherichia coli K-12 genome. We identified all 135 reported terminators and 940 putative terminator sequences beginning no more than 60 nt away from the 3'-end of the annotated transcription units (TU). Putative and reported terminators with the scores above our chosen threshold were found for 37 of the 53 non-coding RNA TU and for almost 50% of the 2592 annotated protein-encoding TU, which correlates well with the number of TU expected to contain rho-independent terminators. We also identified 439 terminators that could function in a bi-directional fashion, servicing one gene on the positive strand and a different gene on the negative strand. Approximately 700 additional termination signals in non-coding regions (NCR) far away from the nearest annotated gene were predicted. This number correlates well with the excess number of predicted 'orphan' promoters in the NCR, and these promoters and terminators may be associated with as yet unidentified TU. The significant number of high scoring hits that occurred within the reading frame of annotated genes suggests that either an additional component of rho-independent terminators exists or that a suppressive mechanism to prevent unwanted termination remains to be discovered.

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/-1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho-dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re-expression (off-on) without affecting the rates of loss of capsule expression (on-off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD-ermC' gene fusions in wild type and Rho-defective Escherichia coli strains. These studies demonstrated that in the Rho-defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off-on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD-ermC' gene fusions in a DNA mismatch-defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo. We show that it is also essential for Nun termination at lambda nutL. Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR. The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL. The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

The Escherichia coli rho-15 mutant (deficient in transcription termination) is known to be incompatible with pBR322 and other plasmids (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We show that failure of pBR322 to transform rho-15 is mediated by transcription from the tet promoter and readthrough from the tet gene into the rom region. Using an isopropyl-beta-D-thiogalactopyranoside-inducible promoter to replace the tet promoter, we have demonstrated that plasmid-specific transcription inhibits growth of the rho-15 host, possibly due to the expression of the Rom protein. The involvement of Rom protein in pBR322-rho-15 incompatibility is further indicated by the following two experiments. (i) Functional inactivation of the rom gene in pBR322 enabled plasmids to transform E. coli rho-15. (ii) Specific overexpression of the rom gene abolished plasmid transformation into E. coli rho-15. An rpoB8(Ts) mutant RNA polymerase which compensated for the termination defect in E. coli rho-15 also restored plasmid-host compatibility, suggesting that Rom-mediated plasmid-host incompatibility is linked to a defect in transcription termination.

The genes required for replication of the temperate bacteriophage P4, which are coded by the phage left operon, are expressed from a constitutive promoter (PLE). In the lysogenic state, repression of the P4 replication genes is achieved by premature transcription termination. The leader region of the left operon encodes all the genetic determinants required for prophage immunity, namely: (i) the P4 immunity factor, a short, stable RNA (CI RNA) that is generated by processing of the leader transcript; (ii) two specific target sequences that exhibit complementarity with the CI RNA. RNA-RNA interactions between the CI RNA and the target sites on the mRNA leader region are essential for transcription termination. To understand how transcription termination is elicited by the P4 immunity mechanism, it is relevant to identify the transcription termination site. This, however, could not be directly inferred from the 3'-end of the transcription products because of the extensive and complex processing and degradation of the leader RNA. In this work, by making use of a tRNA gene as a reporter, we identify the termination site of the immunity transcripts (timm). This is a Rho-dependent terminator located within the first translated gene of the left operon and is regulated by P4 immunity. Analysis of the P4 transcription pattern in Escherichia coli rho mutants suggests that termination at timm may also be important for the efficient processing of the CI RNA.

Escherichia coli RNA polymerase is composed of four different subunits, alpha (present in two copies), beta, beta' and sigma. Among these, the beta' polypeptide shares nine conserved regions with the largest subunits of eukaryotic RNA polymerases, but its role is poorly understood. We isolated novel mutations in a plasmid-borne copy of rpoC, which encodes beta', as dominant suppressors of two temperature-sensitive nusA alleles. All 20 suppressors of nusA11 (single missense mutation) isolated had either of two specific substitutions: Lys for Glu-402 (rpoC10) and Thr for Ala-904 (rpoC111) in the beta' subunit. In vivo and in vitro transcription assays revealed that the rpoC10 allele of beta' participates in Rho-dependent transcription termination. On the other hand, of 20 suppressors of nusA134 (deletion of C-terminal one-third) scattered at 18 distinct sites, 16 were assigned to one of six conserved regions C-I. These results suggested that the conserved domains of the beta' subunit of E. coli RNA polymerase are involved in transcript termination or interaction with termination factor(s).

Rho-dependent terminators constitute one of two major classes of terminators in Escherichia coli. Termination at these sites requires the concerted action of RNA polymerase and rho protein. We present evidence that the efficiency of termination at these sites is governed by kinetic coupling of the rate of transcription of RNA polymerase and the rate of action of rho protein. Termination experiments in vitro indicate that termination efficiency at a rho-dependent terminator is an inverse function of the rate of elongation of RNA polymerase, and each of the mutant phenotypes can be accounted for by the altered rate of elongation of the mutant RNA polymerase. Experiments in vivo show that fast-moving mutant RNA polymerases are termination deficient, while slow-moving mutant RNA polymerases are termination proficient and can suppress the termination deficiency of a slow-acting mutant rho protein. Because of the close coupling of rho action with RNA polymerase, small changes in the elongation rate of RNA polymerase can have very large effects on termination efficiency, providing the cell with a powerful way to modulate termination at rho-dependent terminators.

We have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his-specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho-dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far.

Escherichia coli rho-independent transcription terminators are characterized by an RNA structure having a G+C-rich stem-loop followed by a series of uridine residues, but they can be only partially predicted by the stability of this structure or by its primary sequence. A large number of such terminators have been identified or proposed in the literature, and we have constituted a list of them (148 found in 1021 x 10(3) base-pairs of E. coli DNA sequences) in order to analyze statistically the corresponding RNA hairpins. We show that the size of the loops presents a narrow distribution, that their sequences are not random, and that most loops are closed by a C.G base-pair. In particular, 55% of the loops are tetranucleotides and the most abundant loop sequences are UUCG and GAAA. These loops are abundant in prokaryotic and eukaryotic RNAs, and are known to enhance the stability of RNA hairpins. We propose that these tetraloops play an important role in the nucleation of the nascent RNA structures, as does also the presence of a C.G base-pair closing a hairpin loop. This analysis allows us to propose a model of formation of an RNA hairpin during the termination process and to construct an algorithm of prediction of the terminators in a given DNA sequence. For the E. coli sequences, it clearly distinguishes inter- from intracistronic terminator-like structures, and selects 141 of the 148 rho-independent terminators given in the literature, with a very low background. It also predicts with reasonable accuracy the in vitro termination efficiency of known rho-independent terminators, as well as predicting the existence of 35 as yet uncharacterized terminators.

We have characterized the helicase activity of transcription termination factor rho on a variety of substrates. Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts. Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity. RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho. The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance. Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region. Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream. These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination.

The function of a transcription termination factor, rho, has been studied by using several synthetic DNAs with simple repetitive base sequences as templates for transcription. rho actually exhibits various effects on transcription depending on the base sequence of the template: (1) rho terminates poly(A) synthesis with poly(dA) x poly(dT), poly(dT), or oligo(dT), leading to release of RNA from RNA polymerase. rho also inhibits the synthesis of other homoribopolymers such as poly(U) directed by poly(dA) x poly(dT) and poly(C) and poly(I) directed by poly(dG) x poly(dC), presumably by a similar mechanism. (2) rho inhibits the synthesis of another homoribopolymer, poly(G), directed by poly(dG) x poly(dC) at the step of initiation rather than propagation of transcription. (3) rho stimulates rather than inhibits the synthesis of poly(A-C) and poly(G-U) directed by poly[d(A-C)] x poly[d(G-T)], presumably by enhancing the dissociation of transcription complexes. (4) rho has no influence on the synthesis of poly(A-U) and poly(G-C) directed by poly[d(A-T)] and poly[d(G-C)], respectively. In the first case, but not otherwise, the effect of rho is coupled with its RNA-dependent nucleosidetriphosphate phosphohydrolase activity, as is rho-mediated transcription termination on natural templates. The implication of these results is discussed in reference to the current view that rho acts on transcription complexes that have ceased elongation and causes release of RNA in an energy-requiring reaction.

Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on two structural features of the termination site--a G+C-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these two features are sufficient to specify rho-independent termination in vitro, we have introduced new sequences within a tryptophan (trp) operon structural gene to create two sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. We have compared the activity of our synthetic terminators with those of several termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. These results support a model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript.

Protein factor rho catalyzes site-specific termination of transcription in a reaction requiring hydrolysis of nucleoside triphosphate with eventual release of RNA from RNA polymerase and DNA template. We have characterized the rho-catalyzed RNA release reaction using isolated transcription complexes. Transcription complexes containing T7 D111 DNA, RNA polymerase, and 3H-labeled nascent RNA were formed and isolated by gel filtration on an Agarose 5M column. When the ternary complexes were incubated with rho factor in the presence of ATP, or dATP, significant amounts of nascet RNA were released from the complexes as determined in a membrane filtration assay. Gel electrophoretic analysis of RNA has revealed that rho releases selected species of discrete-sized RNA from among those originally present in the ternary complexes. These results show that rho essentially acts to release RNA from those ternary complexes which have come to pause, and that this reaction proceeds in a discrete step separately from the pausing of RNA synthesis. Under the conditions used, the extent of RNA release widely varied at individual pausing sites and thus the action of rho exhibited certain site-selectivity.

We identified an Escherichia coli gene, designated P14, that is adjacent to and in the opposite orientation to the tonB gene. The 36 base pair intercistronic region between tonB and P14 contains a novel rho-independent transcription terminator that functions bidirectionally, both in vivo and in vitro, to terminate tonB and P14 transcription. Transcription of tonB and P14 terminates at symmetrically equivalent nucleotides, such that the 3' ends of tonB and P14 transcripts are complementary. The terminator is 70% efficient in both directions in vitro. Interestingly, relative rates of in vivo RNA synthesis, immediately prior to and following the terminator, appear to indicate that it is more efficient in the tonB direction (95%) than in the P14 direction (70%). We discuss the possibility that this gene arrangement has regulatory consequences for the expression of tonB.

We have studied the in vitro expression of the osmoregulated proU promoter in Escherichia coli coupled transcription-translation (S-30) extracts as a function of the osmolality of the culture medium used to grow the cells and of the salt concentration added to the extract. These variables represent novel extensions of the use of S-30 extracts to investigate gene regulatory phenomena in vitro. The concentrations of potassium acetate and of the physiologically relevant osmolyte potassium glutamate for maximal expression of the proU promoter are approximately 2-fold higher than the concentrations of these salts providing maximal expression of the lacUV5 promoter. The relative promoter activity of proU with respect to lacUV5 increases more than 30-fold with an increase in salt concentration from 50 to 300 mM. In comparative studies with S-30 extracts prepared from cells grown at high and low osmolalities, we find that at fixed salt concentrations expression of proU is increased 10-fold in the S-30 extract prepared from high osmolality-grown cells, whereas the expression of lacUV5 is increased less than 2-fold. Addition of anti-sigma 70 monoclonal antibodies or purified sigma 70 to the S-30 extract demonstrated that the major proU promoter(s) used in the S-30 extracts is sigma 70-dependent.

Recent studies have suggested that expression of the tryptophanase (tna) operon of Escherichia coli is subject to transcription termination-antitermination control (V. Stewart and C. Yanofsky, J. Bacteriol. 164:731-740, 1985). In vivo studies have indicated that the transcribed leader region, tnaL, contains a site or sites of rho-dependent transcription termination (rho is the polypeptide product of the gene rho). We now report direct in vitro evidence that tnaL contains rho-dependent termination sites. In vivo termination appeared to occur at the rho-dependent termination sites identified in vitro. Transcription pausing analyses correlated sites of pausing in tnaL with sites of rho-dependent termination.

Transcription antitermination by phage lambda N protein is reproduced in vitro solely with purified components. We have placed a strong rho-independent terminator, lambda tR', in the PL operon about 200 base pairs downstream from the N-recognition site, nutL, and have monitored terminated and run-off transcripts produced by single-round transcription of linear plasmids. In the presence of NusA, one of several host factors implicated in antitermination, N is found to virtually abolish termination at tR'. N is unable to suppress termination if the terminator is preceded by a defective nut site. Thus, during transcription through the nut site, N and NusA can modify RNA polymerase to a termination-resistant form in the absence of any other accessory factor.

The genes specifying chemotaxis, motility, and flagellar function in Escherichia coli are coordinately regulated and form a large and complex regulon. Despite the importance of these genes in controlling bacterial behavior, little is known of the molecular mechanisms that regulate their expression. We have identified a minor form of E. coli RNA polymerase that specifically transcribes several E. coli chemotaxis/flagellar genes in vitro and is likely to carry out transcription of these genes in vivo. The enzyme was purified to near homogeneity based on its ability to initiate transcription of the E. coli tar chemotaxis gene at start sites that are used in vivo. Specific tar transcription activity is associated with a polypeptide of apparent 28-kDa molecular mass that remains bound to the E. coli RNA polymerase throughout purification. This peptide behaves as a secondary sigma factor--designated sigma F--because it restores specific tar transcription activity when added to core RNA polymerase. The sigma F holoenzyme also transcribes the E. coli tsr and flaAI genes in vitro as well as several Bacillus subtilis genes that are transcribed specifically by the sigma 28 form of B. subtilis RNA polymerase. The latter holoenzyme is implicated in transcription of flagellar and chemotaxis genes in B. subtilis. Hence E. coli sigma F holoenzyme appears to be analogous to the B. subtilis sigma 28 RNA polymerase, both in its promoter specificity and in the nature of the regulon it controls.

Promoters located within the Tn10 insertion element cause transcription of "host" sequences adjacent to both ends of the inserted Tn10 element. These promoters are usually not observed in genetic experiments because their transcripts are efficiently terminated at nearby rho-dependent termination sites. The observations presented here provide an explanation for several confusing aspects of transposon behavior and suggest the possibility that many transposons possess promoters that have escaped detection for similar reasons.

The bacteriophage lambda N gene product positively controls development by preventing termination of transcription at terminator sites critical to the sequential expression of phage genes. Many host transcription factors, including RNA polymerase, are involved in N gene action. Recent findings have shown that ribosomal proteins are also involved. The current understanding of how the N protein affects transcription termination is reviewed, and a possible model and current problems are discussed.

It has been generally accepted that transcriptional polarity in prokaryotic systems is due to an uncoupling of translation and transcription which unmasks latent rho-dependent termination sites in a polycistronic messenger RNA. In this report, we identify and characterize rho-dependent termination sites responsible for transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12. The ilvG gene in the wild-type E. coli K12 ilvGMEDA operon contains a frameshift site which results in termination of translation in the middle of the gene. Mutations have been characterized which restore the reading frame of this gene. In addition to allowing full-length expression of the ilvG product, these mutations cause a 3-4-fold elevation in the expression of the operon distal genes. This transcriptional polarity effect on operon distal genes also has been shown to be relieved by rho suppressor mutations. We have used in vitro transcription experiments to identify rho-dependent transcriptional termination sites downstream of the frameshift site in the ilvG gene. Three tandem rho-dependent sites have been located in the ilv'GM' gene region using transcription reactions containing linear or supercoiled plasmid DNA templates. Accumulatively, these rho-dependent termination sites account for about 80% in vitro transcription termination, which is in agreement with the in vivo measurements of transcriptional polarity on operon distal gene expression. These transcriptional experiments provide in vitro confirmation for the latent rho-dependent termination site model of transcriptional polarity.

To determine whether E. coli rho protein mediates termination of transcription by interacting with specific segments of the nascent transcript, DNA oligonucleotides were used to sequester segments of phage lambda cro mRNA in hybrid helices. Formation of hybrids was demonstrated with RNAase H assays. Oligonucleotides complementary to either of two distinct, single-stranded sequences near the 3' end inhibited rho action at tR1, while oligonucleotides complementary to the sequence between those segments or to more 5' segments did not. The inhibitory oligonucleotides did not affect the elongation of cro mRNA or rho action on other transcripts. The results indicate that termination of transcription at tR1 is dependent upon contact of rho factor with specific, single-stranded domains near the 3' end of cro mRNA.

The nusA1 and nusB5 mutations in a partial suppression of polarity and thus transcription termination in Escherichia coli. As these mutations block the transcription antitermination activity of bacteriophage lambda N gene product, they paradoxically seem to enhance transcription termination at phage termination sites. The rho mutation HDF026 displays almost identical properties. The observations suggest that the nusA and nusB gene products may act as termination factors analogous to rho protein.

A 178-bp repeat sequence comprises the distal end of the tyrT operon and also encodes the signal for Rho-dependent transcription termination. It is shown here that Rho-dependent transcription termination is highly efficient for each of the individually cloned first and second repeats. The presence of either repeat results in a termination efficiency of 88% (12% readthrough). When an individual DNA repeat is shortened, starting from the promoter-proximal end, so that only 56 bp remain upstream from the termination site, Rho-dependent termination activity is reduced approximately three-fold (to give 38% readthrough) and with further shortening, so that only 15 bp remain upstream, Rho-dependent termination is reduced an additional 1.8-fold (to give 59% readthrough). Finally, it is shown that the presence or absence of the tyrT terminator upstream from the lambda tR1 terminator does not affect the efficiency of transcription at termination lambda tr1.

We examined the in vitro concentration dependence of the effects of Nus A on transcription termination and pausing to determine if Nus A affects both pausing and termination in vitro by binding to a single site on the transcription complex. Nus A was shown to cause maximal increases of pausing at a concentration approximately equimolar to RNA polymerase. However, the effects of Nus A on termination require much higher Nus A concentrations than are required for pausing. It is therefore likely that the effects of Nus A on pausing and termination result from the binding of Nus A to different sites on the transcription complex. Since proteins that probably bind RNA nonspecifically were also shown to strongly reduce termination at a Rho-dependent terminator, Nus A may decrease Rho-dependent termination by binding nonspecifically to RNA. This proposal is consistent with most of the available data on the in vitro effects of Nus A and provides a mechanistic basis for previously unexplained details of Nus A caused decreases in Rho-dependent termination. We further speculate that most or all of the in vivo roles of Nus A may involve the enhancement of pausing.