Factor-dependent Transcription Termination Research Articles

Single-molecule characterization of Sen1 translocation properties provides insights into eukaryotic factor-dependent transcription termination.

Sen1 is an essential helicase for factor-dependent transcription termination in Saccharomyces cerevisiae, whose molecular-motor mechanism has not been well addressed. Here, we use single-molecule experimentation to better understand the molecular-motor determinants of its action on RNA polymerase II (Pol II) complex. We quantify Sen1 translocation activity on single-stranded DNA (ssDNA), finding elevated translocation rates, high levels of processivity and ATP affinities. Upon deleting the N- and C-terminal domains, or further deleting different parts of the prong subdomain, which is an essential element for transcription termination, Sen1 displays changes in its translocation properties, such as slightly reduced translocation processivities, enhanced translocation rates and statistically identical ATP affinities. Although these parameters fulfil the requirements for Sen1 translocating along the RNA transcript to catch up with a stalled Pol II complex, we observe significant reductions in the termination efficiencies as well as the factions of the formation of the previously described topological intermediate prior to termination, suggesting that the prong may preserve an interaction with Pol II complex during factor-dependent termination. Our results underscore a more detailed rho-like mechanism of Sen1 and a critical interaction between Sen1 and Pol II complex for factor-dependent transcription termination in eukaryotes.

Structural basis of Rho-dependent transcription termination.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli1-4. Here we report the preparation of complexes that arefunctional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopystructures of thecomplexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer;have RNA threaded through the central channel of Rho;have 60 nucleotidesof RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotidesof RNA interacting sequence-specifically with the central channel of Rho;have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP;and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

Quantitative characterization of gene regulation by Rho dependent transcription termination

Rho factor dependent transcription termination (RTT) is common within the coding sequences of bacterial genes and it acts to couple transcription and translation levels. Despite the importance of RTT for gene regulation, its effects on mRNA and protein concentrations have not been quantitatively characterized. Here we demonstrate that the exogenous cfp gene encoding the cyan fluorescent protein can serve as a model for gene regulation by RTT. This was confirmed by showing that Psu and bicyclomycin decrease RTT and increase full length cfp mRNAs (but remarkably they have little effect on protein production). We then use cfp to characterize the relationship between its protein and full length mRNA concentrations when the translation initiation rate is varied by sequence modifications of the translation initiation region (TIR). These experiments reveal that the fold change in protein concentration (RP) and the fold change in full length mRNA concentration (Rm) have the relationship RP≈Rmb, where b is a constant. The average value of b was determined from three separate data sets to be ~3.6. We demonstrate that the above power law function can predict how altering the translation initiation rate of a gene in an operon will affect the mRNA concentrations of downstream genes and specify a lower bound for the associated changes in protein concentrations. In summary, this study defines a simple phenomenological model to help program expression from single genes and operons that are regulated by RTT, and to guide molecular models of RTT.

Inhibition of factor-dependent transcription termination in Escherichia coli might relieve xenogene silencing by abrogating H-NS-DNA interactions in vivo

Many horizontally acquired genes (xenogenes) in the bacterium Escherichia coli are maintained in a silent transcriptional state by the nucleoid-associated transcription regulatory protein H-NS. Recent evidence has shown that antibiotic-mediated inhibition of the transcription terminator protein Rho leads to de-repression of horizontally acquired genes, akin to a deletion of hns. The mechanism behind this similarity in outcomes between the perturbations of two distinct processes remains unclear. Using ChIP-seq of H-NS in wild-type cells, in addition to that in cells treated with bicyclomycin--a specific inhibitor of Rho, we show that bicyclomycin treatment leads to a decrease in binding signal for H-NS to the E. coli chromosome. Rho inhibition leads to RNA polymerase readthrough, which in principle could displace H-NS from the DNA, thus leading to transcriptional derepression of H-NS-silenced genes. Other possible mediators of the effect of Rho on H-NS are discussed. A possible positive feedback between Rho and H-NS might help reinforce xenogene silencing.

Rho-dependent transcription termination is essential to prevent excessive genome-wide R-loops in Escherichia coli

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translation-uncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.

Compromised Factor-Dependent Transcription Termination in a nusA Mutant of Escherichia coli: Spectrum of Termination Efficiencies Generated by Perturbations of Rho, NusG, NusA, and H-NS Family Proteins

The proteins NusA and NusG, which are essential for the viability of wild-type Escherichia coli, participate in various postinitiation steps of transcription including elongation, antitermination, and termination. NusG is required, along with the essential Rho protein, for factor-dependent transcription termination (also referred to as polarity), but the role of NusA is less clear, with conflicting reports that it both promotes and inhibits the process. In this study, we found that a recessive missense nusA mutant [nusA(R258C)] exhibits a transcription termination-defective (that is, polarity-relieved) phenotype, much like missense mutants in rho or nusG, but is unaffected for either the rate of transcription elongation or antitermination in λ phage. Various combinations of the rho, nusG, and nusA mutations were synthetically lethal, and the lethality was suppressed by expression of the N-terminal half of nucleoid protein H-NS. Our results suggest that NusA function is indeed needed for factor-dependent transcription termination and that an entire spectrum of termination efficiencies can be generated by perturbations of the Rho, NusG, NusA, and H-NS family of proteins, with the corresponding phenotypes extending from polarity through polarity relief to lethality.

Host Factor Titration by Chromosomal R-loops as a Mechanism for Runaway Plasmid Replication in Transcription Termination-defective Mutants of Escherichia coli

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 °C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 °C following overexpression of rnhA +. Mutant combinations rnhA- nusG or recG- rho were synthetically lethal at 21 °C, with the former being suppressed by recG + overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 °C or 37 °C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA + or recG +. Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom +, and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and Δ64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication.

Elongation properties of vaccinia virus RNA polymerase: pausing, slippage, 3' end addition, and termination site choice.

We have analyzed the elongation properties of vaccinia virus RNA polymerase during a single round of transcription in vitro. RNA-labeled ternary complexes were halted at a unique template position located upstream of a T-run (TTTTTTTTT) in the nontemplate strand; this element encodes an RNA signal for factor-dependent transcription termination at distal sites on the template. The halted ternary complexes were purified and allowed to resume elongation under a variety of conditions. We found that the T-run constituted a strong elongation block, even at high nucleotide concentrations. The principal sites of pausing were at a C position situated two nucleotides upstream of the first T in the T-run and at the first three to four T positions within the T-run. There was relatively little pausing at the five downstream Ts. Intrinsic pausing was exacerbated at suboptimal nucleotide concentrations. Ternary complexes arrested by the T-run at 10 microM NTPs rapidly traversed the T-run when the NTP pool was increased to 1 mM. Limiting GTP (1 microM) resulted in polymerase stuttering at the 3' margin of the T-run, immediately prior to a templated G position; this generated a ladder of slippage synthesis products. We found that vaccinia ternary complexes remained intact after elongating to the very end of a linear DNA template and that such complexes catalyzed the addition of extra nucleotides to the 3' end of the RNA chain. The 3' end addition required much higher concentrations of NTPs than did templated chain elongation. Finally, we report that factor-dependent transcription termination by vaccinia RNA polymerase downstream of the T-run was affected by nucleotide concentration. Limiting UTP caused the polymerase to terminate at sites closer to the UUUUUNU termination signal. This is consistent with the kinetic coupling model for factor-dependent termination.

An ATPase Component of the Transcription Elongation Complex Is Required for Factor-dependent Transcription Termination by Vaccinia RNA Polymerase

Vaccinia virus RNA polymerase terminates transcription in response to a specific signal UUUUUNU in the nascent transcript. Transduction of this signal to the elongating polymerase requires a virus-encoded termination factor, VTF. The existence of a second termination factor was suggested by the finding that transient exposure of purified elongation complexes to heparin rendered them refractory to VTF-induced termination. Loss of termination competence correlated with the removal of several polypeptide components of the elongation complex. We present the identification of factor X, an activity that restored VTF responsiveness to heparin-stripped ternary complexes. We propose that factor X, which has an associated DNA-dependent ATPase activity, mediates the requirement for ATP hydrolysis during transcription termination.

Factor-dependent Release of Nascent RNA by Ternary Complexes of Vaccinia RNA Polymerase

Factor-dependent transcription termination during synthesis of vaccinia early mRNAs occurs at heterogeneous sites downstream of a UUUUUNU signal in the nascent transcript. The choice of termination site is flexible and is determined by a kinetic balance between nascent chain elongation and the transmission of the RNA signal to the polymerase. To eliminate ongoing elongation as a variable, we have established a system to study transcript release by purified ternary complexes halted at a defined template position 50-nucleotides 3' of the first U residue of the termination signal. Release of the nascent RNA depends on the vaccinia termination factor (VTF) and an ATP cofactor. Transcript release is blocked by BrUMP substitution within the termination signal of the nascent RNA. In these respects, the release reaction faithfully mimics the properties of the termination event. We demonstrate that ternary complexes are refractory to VTF-mediated transcript release when the first U of the UUUUUNU signal is situated 20 nucleotides from the growing point of the nascent chain. Ribonuclease footprinting of the arrested ternary complexes defines a nascent RNA binding site on the polymerase elongation complex that encompasses a 16-21 nucleotide RNA segment extending proximally from the 3' end of the chain. We surmise that access of VTF to the signal sequence is prevented when UUUUUNU is bound within the nascent RNA binding site. Hence, physical not kinetic constraints determine the minimal distance between the signal and potential sites of 3' end formation.

Factor-dependent transcription termination by vaccinia RNA polymerase. Kinetic coupling and requirement for ATP hydrolysis

Transcription termination during synthesis of vaccinia early mRNAs occurs downstream of a UUUUUNU signal in the nascent transcript and requires a virus-encoded termination factor (VTF), which is identical with the vaccinia mRNA capping enzyme. Using purified transcription complexes halted at defined sites on linear DNA templates, we have examined the order and timing of events during a single round of elongation and termination. We find that although cap synthesis occurs by the time the nascent RNA is 31 nucleotides long, capping enzyme is not stably associated with the elongation complex at this stage. Stable interaction, defined by the formation of a termination-competent complex, requires a longer nascent RNA, e.g. 51 nucleotides, but does not depend on prior transcription of the termination signal. The acquisition of termination competence correlates temporally with the physical association of capping enzyme/VTF with the elongation complex, as revealed by UV cross-linking of the capping enzyme large subunit to the nascent RNA chain. Subsequent induction of termination and transcript release by capping enzyme requires energy, specifically the hydrolysis of ATP. The choice of termination site is flexible and is determined by a kinetic balance between the rate of polymerase elongation and the rate of signaling. Signaling rate is related directly to the concentration of hydrolyzable ATP. An apparent lower limit of 33 nucleotides between the 5' boundary of the termination signal and the most proximal termination site implies that the UUUUUNU signal must be extruded from the RNA polymerase before it can be acted upon by VTF. Similarities between VTF-dependent termination and rho-dependent termination underscore an evolutionarily conserved mechanism for RNA signal transduction to the elongating RNA polymerase.

Pleiotropic effects of the rpoC10 mutation affecting the RNA polymerase β′ subunit of Escherichia coli on factor‐dependent transcription termination and antitermination

Escherichia coli RNA polymerase is composed of four different subunits, 2 alpha, beta, beta' and sigma. Among these subunits, the role of beta' is poorly understood. The rpoC10 mutation affecting beta' has been isolated as a suppressor mutation of the temperature-sensitive nusA11 mutant. DNA sequence analysis revealed that the rpoC10 mutant is a substitution of Lys for Glu-402. This increased positive charge appears to compensate for the increased negative charge present in the nusA11 protein (Asp for Gly-181). In vivo measurements of reporter gene expression have revealed that rpoC10 restores rho-dependent termination but fails to restore rho-independent termination in nusA11. Moreover, the rpoC10 mutation, in combination with any nusA mutation, inhibited lambda Q-mediated antitermination without affecting N antitermination and severely restricted lambda phage development. The inhibition of Q function and lambda growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase beta' subunit plays a crucial role in factor-dependent transcription termination and antitermination.

Requirement for E. coli NusG protein in factor-dependent transcription termination

The 21 kd NusG protein is essential for E. coli viability. Cells depleted for NusG were defective for factor-dependent transcription termination. Rho-induced polarity in the gal operon and the Rho-dependent λtR1 and λtL1 terminators were suppressed in NusG-deficient cells. NusG depletion inactivated the phage HK022 Nun termination factor. In contrast, the factor-independent λtl terminator was fully active in NusG-depleted cells and could be suppressed by phage λN function.

Altered Expression of the Bacteriophage T4 Gene 41 (Primase-Helicase) in an Escherichia coli rho Mutant

Bacteriophage T4 gene 41 protein is an essential replication protein, part of the primase-helicase required for lagging strand DNA synthesis. In a T4+ infection, 41 RNA is first expressed as a polycistronic transcript attached to the upstream RNA of genes uvsX (recombination protein) and 40 (stimulates head formation (Hinton, D. M. (1989) J. Biol. Chem. 264, 14432-14439). As infection proceeds, less of the upstream RNA extends into gene 41 due to an RNA 3' end, approximately equal to 60 bases downstream of uvsX. DNA sequence analysis of this region positions this end within gene 40, immediately after a GC-rich hairpin. This end probably arises from host factor-dependent transcription termination or RNA processing since it is observed in RNA expressed by a uvsX-40-41 plasmid in vivo, but is not seen after in vitro transcription with purified Escherichia coli RNA polymerase. The E. coli transcription termination (rho) mutant rho026 has been characterized as a rho mutation whose terminating activity is not effectively overcome by phage lambda antitermination (Das, A., Gottesman, M. E., Wardwell, J., Trisler, P., and Gottesman, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5530-5534). During a T4+ abortive infection of rho026, the levels of some phage proteins, including 41, are depressed; a T4 phage mutant in goF gives wild type protein patterns in rho026 (Stitt, B. L., and Mosig, G. (1989) J. Bacteriol., in press). The RNA analyses presented here demonstrate that the severalfold decrease in 41 protein in rho026 is accompanied by a similar decrease in 41 RNA. There is both a general reduction in polycistronic uvsX-40-41 RNA and a 2-2.5-fold increase in the proportion of uvsX RNA ending at the 3' end. Infection of rho026 by T4 goF1 returns the relative amount of RNA reading into 41 versus that stopped to near a wild type level. These results suggest that host rho and the T4 goF are involved in the expression of T4 41 RNA.

Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA.

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.

Rho Factor-dependent transcription termination: Interference by a mutant rho

The rho protein isolated from a strain of Escherichia coli with the rho1 ( suA1) mutant allele is defective in interactions with RNA that are coupled to ATP hydrolysis. Here we show that the rho1 allele is partially dominant over wild-type and demonstrate that the mechanism of that dominance is due to an interference of wild-type rho factor function by the defective rho factor. The rho1 mutant protein can inhibit transcription termination and RNA-dependent ATPase activities of normal rho protein. Inhibition of the ATPase activity with excess RNA occurs by exchange of subunits to form hybrid hexamers in which the defective subunits apparently disrupt co-operative interactions essential for wild-type subunit function.