Mechanism Of Transcription Termination Research Articles

Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch

Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswicthes, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg2+ ions, which in turn are octahedrally coordinated to waters and five inwardly-pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state defines how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch most likely functions in gene regulation through a transcription termination mechanism.

Problem‐solving test: The mechanism of transcription termination by the rho factor

Terms to be familiar with before starting to solve the test: Transcription termination, recombinant plasmid, annealing, molecular hybridization, heat denaturation, agarose gel electrophoresis, autoradiography, RNA polymerase, and antiparallel orientation.

An allosteric mechanism of Rho-dependent transcription termination

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria. Although Rho was the first termination factor to be discovered, the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle; that is, it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP-Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allosterically induced rearrangement of the RNAP catalytic centre by means of the evolutionarily conserved mobile trigger-loop domain, which is also required for EC dissociation. The key structural and functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution.

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3' end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF.

A Novel Tandem Reporter Quantifies RNA Polymerase II Termination in Mammalian Cells

BackgroundMaking the correct choice between transcription elongation and transcription termination is essential to the function of RNA polymerase II, and fundamental to gene expression. This choice can be influenced by factors modifying the transcription complex, factors modifying chromatin, or signals mediated by the template or transcript. To aid in the study of transcription elongation and termination we have developed a transcription elongation reporter system that consists of tandem luciferase reporters flanking a test sequence of interest. The ratio of expression from the reporters provides a measure of the relative rates of successful elongation through the intervening sequence.Methodology/Principal FindingsSize matched fragments containing the polyadenylation signal of the human β-actin gene (ACTB) and the human β-globin gene (HBB) were evaluated for transcription termination using this new ratiometric tandem reporter assay. Constructs bearing just 200 base pairs on either side of the consensus poly(A) addition site terminated 98% and 86% of transcription for ACTB and HBB sequences, respectively. The nearly 10-fold difference in read-through transcription between the two short poly(A) regions was eclipsed when additional downstream poly(A) sequence was included for each gene. Both poly(A) regions proved very effective at termination when 1100 base pairs were included, stopping 99.6% of transcription. To determine if part of the increased termination was simply due to the increased template length, we inserted several kilobases of heterologous coding sequence downstream of each poly(A) region test fragment. Unexpectedly, the additional length reduced the effectiveness of termination of HBB sequences 2-fold and of ACTB sequences 3- to 5-fold.Conclusions/SignificanceThe tandem construct provides a sensitive measure of transcription termination in human cells. Decreased Xrn2 or Senataxin levels produced only a modest release from termination. Our data support overlap in allosteric and torpedo mechanisms of transcription termination and suggest that efficient termination is ensured by redundancy.

Single Molecule Studies Reveal the Mechanism and Energetics of Transcriptional Termination

Intrinsic transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a G:C‐rich RNA hairpin followed by a 7–9 nt, U‐rich tract. We used single‐molecule optical trapping techniques to investigate the mechanism by which elements from three representative terminators (his, t500, tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias DNA translocation did not affect termination efficiency (TE). However, the force‐dependent kinetics of release at the t500 terminator and the force‐dependent TE of a mutant imply a forward translocation mechanism for this terminator. Tension applied to isolated U‐tracts induced transcript release in a manner consistent with shearing of the RNA:DNA hybrid. We deduce that different mechanisms of EC dissociation, involving hypertranslocation or RNA:DNA shearing, operate at terminators with different U‐tract sequences. Tension applied to RNA at full terminators suggests that closure of the final 2–3 hairpin bases supplies the energy that destabilizes the hybrid, and that competing nascent RNA structures modulate TE by slowing terminator hairpin formation. We propose a quantitative model based on energetics that successfully predicts the behavior for all three terminators and several mutant variants.

Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea

The basal transcription apparatus of archaea is well characterized. However, much less is known about the mechanisms of transcription termination and translation initation. Recently, experimental determination of the 5′-ends of ten transcripts from Pyrobaculum aerophilum revealed that these are devoid of a 5′-UTR. Bioinformatic analysis indicated that many transcripts of other archaeal species might also be leaderless. The 5′-ends and 3′-ends of 40 transcripts of two haloarchaeal species, Halobacterium salinarum and Haloferax volcanii, have been determined. They were used to characterize the lengths of 5′-UTRs and 3′-UTRs and to deduce consensus sequence-elements for transcription and translation. The experimental approach was complemented with a bioinformatics analysis of the H. salinarum genome sequence. Furthermore, the influence of selected 5′-UTRs and 3′-UTRs on transcript stability and translational efficiency in vivo was characterized using a newly established reporter gene system, gene fusions, and real-time PCR. Consensus sequences for basal promoter elements could be refined and a novel element was discovered. A consensus motif probably important for transcriptional termination was established. All 40 haloarchaeal transcripts analyzed had a 3′-UTR (average size 57 nt), and their 3′-ends were not posttranscriptionally modified. Experimental data and genome analyses revealed that the majority of haloarchaeal transcripts are leaderless, indicating that this is the predominant mode for translation initiation in haloarchaea. Surprisingly, the 5′-UTRs of most leadered transcripts did not contain a Shine-Dalgarno (SD) sequence. A genome analysis indicated that less than 10% of all genes are preceded by a SD sequence and even most proximal genes in operons lack a SD sequence. Seven different leadered transcripts devoid of a SD sequence were efficiently translated in vivo, including artificial 5′-UTRs of random sequences. Thus, an interaction of the 5′-UTRs of these leadered transcripts with the 16S rRNA could be excluded. Taken together, either a scanning mechanism similar to the mechanism of translation initiation operating in eukaryotes or a novel mechanism must operate on most leadered haloarchaeal transcripts.

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

A recent model for the mechanism of intrinsic transcription termination involves dissociation of the RNA from forward-translocated (hypertranslocated) states of the complex [Yarnell WS, Roberts JW (1999) Science, 284:611-615]. The current study demonstrates that halted elongation complexes of T7 RNA polymerase in the absence of termination signals can also dissociate via a forward-translocation mechanism. Shortening of the downstream DNA or the introduction of a stretch of mismatched DNA immediately downstream of the halt site reduces a barrier to forward translocation and correspondingly reduces the lifetime of halted complexes. Conversely, introduction of a cross-link downstream of the halt site increases the same barrier and leads to an increase in complex lifetime. Introduction of a mismatch within the bubble reduces a driving force for forward translocation and correspondingly increases the lifetime of the complex, but only for mismatches at the upstream edge of the bubble, as predicted by the model. Mismatching only the two most upstream of the eight bases in the bubble provides a maximal increase in complex stability, suggesting that dissociation occurs primarily from early forward-translocated states. Finally, addition in trans of an oligonucleotide complementary to the nascent RNA just beyond the hybrid complements the loss of driving force derived from placement of a mismatch within the bubble, confirming the expected additivity of effects. Thus, forward translocation is likely a general mechanism for dissociation of elongation complexes, both in the presence and absence of intrinsic termination signals.

Expression of thebmpBGene ofBorrelia burgdorferiIs Modulated by Two Distinct Transcription Termination Events

bmp gene family 36 of Borrelia burgdorferi, the agent of Lyme disease, comprises four paralogs: bmpA, bmpB, bmpC, and bmpD. The bmpA and bmpB genes constitute an operon. All four genes have been found to be transcribed in cultured spirochetes. Expression from the bmpAB operon results in three distinct transcripts of 1.1, 1.6, and 2.4 kb, and the relative expression of bmpA mRNA is three- to fourfold greater than that of bmpB mRNA. However, thus far only expression of the BmpA protein has been demonstrated. Therefore, in this study we characterized the origins of the three transcripts and compared the relative expression of the BmpA and BmpB proteins. Northern blotting revealed that the three distinct transcripts originated from a single promoter located upstream of bmpA but terminated either 3' to the bmpA (1.1-kb RNA) or bmpB (2.4-kb RNA) gene or, most unusually, within the bmpB gene (1.6-kb RNA). Termination within the bmpB gene was associated with a functional Rho-independent transcription terminator. At the protein level, we also observed a 4.3-fold greater abundance of BmpA compared to that of BmpB. These studies identify a transcription termination mechanism in B. burgdorferi resulting in the disparate expression of the two genes of the bmpAB operon.

The Speed of RNA Transcription and Metabolite Binding Kinetics Operate an FMN Riboswitch

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

The Purine Efflux Pump PbuE in Bacillus subtilis Modulates Expression of the PurR and G-Box (XptR) Regulons by Adjusting the Purine Base Pool Size

In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP). The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP. The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism. The G-box regulon effector molecules are hypoxanthine and guanine. pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon. The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine. Here we show that the expression of pbuE'-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine. Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs. In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased. Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

Influence of substrate composition on the helicase activity of transcription termination factor Rho: reduced processivity of Rho hexamers during unwinding of RNA-DNA hybrid regions.

Transcription termination factor Rho forms ring-shaped hexameric structures that load onto segments of the nascent RNA transcript that are C-rich and mostly single-stranded. This interaction converts Rho hexamers into active molecular motors that use the energy resulting from their ATP hydrolase activity to move towards the transcript 3'-end. Upon translocation along the RNA chain, Rho can displace physical roadblocks, such as those formed by RNA-DNA helices, a feature that is likely central to the transcription termination mechanism. To study this "translocase" (helicase) activity, we have designed a collection of Rho substrate chimeras containing an RNA-DNA helix located at various positions with respect to a short (47 nucleotides) artificial loading site. We show that these synthetic constructs represent interesting model substrates able to engage in a productive interaction with Rho and to direct NTP-dependent [5'-->3']-translocation of the hexamers. Using both single and multiple-cycle experimental set-ups, we have also found that Rho helicase activity is strongly dependent on the substrate composition and reaction conditions. For this reason, the rate-limiting step of the helicase reaction could not be identified unambiguously. Yet, the linear dependence of the reaction rate on the hybrid length suggests that helicase action on the RNA-DNA region could be controlled by a unique slow step such as Rho activation, conformational rearrangement, or DNA release. Moreover, removal of the DNA strand occurred at a significant cost for the Rho enzyme, inducing, on average, dissociation from the substrate for every 60-80 base-pairs of hybrid unwound. These results are discussed in relation to the known requirements for Rho substrates, general features of hexameric helicases, and current models for Rho-dependent transcription termination.

Definition of a Second Bacillus subtilis pur Regulon Comprising the pur and xpt-pbuX Operons plus pbuG , nupG ( yxjA ), and pbuE ( ydhL )

In Bacillus subtilis expression of genes or operons encoding enzymes and other proteins involved in purine synthesis is affected by purine bases and nucleosides in the growth medium. The genes belonging to the PurR regulon (purR, purA, glyA, guaC, pbuO, pbuG, and the pur, yqhZ-folD, and xpt-pbuX operons) are controlled by the PurR repressor, which inhibits transcription initiation. Other genes are regulated by a less-well-described transcription termination mechanism that responds to the presence of hypoxanthine and guanine. The pur operon and the xpt-pbuX operon, which were studied here, are regulated by both mechanisms. We isolated two mutants resistant to 2-fluoroadenine in which the pur operon and the xpt-pbuX operon are expressed at increased levels in a PurR-independent manner. The mutations were caused by deletions that disrupted a potential transcription terminator structure located immediately upstream of the ydhL gene. The 5' part of the ydhL leader region contained a 63-nucleotide (nt) sequence very similar to the 5' ends of the leaders of the pur and xpt-pbuX operons. Transcripts of these regions may form a common tandem stem-loop secondary structure. Two additional genes with potential leader regions containing the 63-nt sequence are pbuG, encoding a hypoxanthine-guanine transporter, and yxjA, which was shown to encode a purine nucleoside transporter and is renamed nupG. Transcriptional lacZ fusions and mutations in the 63-nt sequence encoding the possible secondary structures provided evidence that expression of the pur and xpt-pbuX operons and expression of the ydhL, nupG, and pbuG genes are regulated by a common mechanism. The new pur regulon is designated the XptR regulon. Except for ydhL, the operons and genes were negatively regulated by hypoxanthine and guanine. ydhL was positively regulated. The derived amino acid sequence encoded by ydhL (now called pbuE) is similar to the amino acid sequences of metabolite efflux pumps. When overexpressed, PbuE lowers the sensitivity to purine analogs. Indirect evidence indicated that PbuE decreases the size of the internal pool of hypoxanthine. This explains why the hypoxanthine- and guanine-regulated genes are expressed at elevated levels in a mutant that overexpresses pbuE.

Analysis of the Intrinsic Transcription Termination Mechanism and Its Control

Publisher Summary This chapter describes approaches that permit dissecting the termination process in several steps and identifying individual roles of the terminator elements, the hairpin and T-stretch, in each step. To study the basic mechanism of intrinsic termination and its control, an in vitro reconstituted system is used that contains Escherichia coli (E.coli) RNAP, a linear DNA template with a strong promoter and the intrinsic terminator, and pure elongation factors, NusA and N. E. coli NusA protein works as a general termination factor, which significantly increases the efficiency of many intrinsic terminators in vitro and in vivo. The trapped complex (TC) represents a unique configuration of the elongation complexes (EC) that occurs exclusively at the intrinsic termination points. Pausing at the tR2 termination point is determined by the downstream portion of its T-stretch. Pausing provides an additional time for the hairpin to form and is required for efficient termination under physiological conditions. To exclude the kinetic component (pausing) from the analysis of termination and to study the “mechanistic” component of the termination process in real time, the slow termination approach has been developed. This approach utilizes a modified A1-tR2 template carrying point substitutions within the T-stretch, rendering the RNA: DNA hybrid strong enough to delay the hairpin folding.

Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri.

Bacteriophage SfV is a temperate serotype-converting phage of Shigella flexneri. SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage lambda, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that orf-41 encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two S. flexneri strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has cos sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of Escherichia coli O157:H7 and Haemophilus influenzae. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the E. coli K-12 genome were noted, suggesting that these three phages have common evolutionary origins.

Polymerase slippage at vesicular stomatitis virus gene junctions to generate poly(A) is regulated by the upstream 3'-AUAC-5' tetranucleotide: implications for the mechanism of transcription termination.

Termination of mRNA synthesis in vesicular stomatitis virus (VSV), the prototypic rhabdovirus, is controlled by a 13-nucleotide gene end sequence which comprises the conserved tetranucleotide 3'-AUAC-5', the U(7) tract and the intergenic dinucleotide. mRNAs terminated at this sequence possess 100- to 300-nucleotide-long 3' poly(A) tails which are thought to result from polymerase slippage (reiterative transcription) by the VSV polymerase on the U(7) tract. Previously we determined that in addition to the AUAC tetranucleotide, the U(7) tract was an essential signal in the termination process. Shortening or interrupting the U(7) tract abolished termination. These altered U tracts also prevented the polymerase from performing reiterative transcription necessary for generation of the mRNA poly(A) tail and thus established seven residues as the minimum length of U tract that allowed reiterative transcription to occur. In this study we investigated whether sequences other than the essential U(7) tract are involved in controlling polymerase slippage. We investigated whether the AUAC tetranucleotide affected the process of reiterative transcription by analyzing the nucleotide sequence of RNAs transcribed from altered subgenomic templates and infectious VSV variants. The tetranucleotide was found to regulate reiterative transcription on the U(7) tract. The extent of polymerase slippage was governed not by specific tetranucleotide sequences but rather by nucleotide composition such that slippage occurred when the tetranucleotide was composed of A or U residues but not when it was composed of G or C residues. This suggested that polymerase slippage was controlled, at least in part, by the strength of base pairing between the template and nascent strands. Further data presented here indicate that the tetranucleotide contains both a signal that directs the VSV polymerase to slip on the downstream U(7) tract and also a signal that directs a slipping polymerase to terminate mRNA synthesis.

Transcriptional termination and coupled polyadenylation in vitro.

Using a coupled, in vitro transcription and polyadenylation system we have investigated the molecular mechanism of transcriptional termination by RNA polymerase II (PolII). We showed previously that specific G-rich sequences pause transcription and then activate polyadenylation. We show that physiological pause sites activate polyadenylation in our in vitro system. We also investigate the mechanism of PolII transcriptional termination, and show that these transcripts are either directly released from the transcription complex or are 3' end processed while still attached to the complex. We also show that 3' product (generated by cleavage/polyadenylation) remains associated with the transcription complex, but is rapidly degraded on it.

Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules.

Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro. An RNAP molecule remains at a canonical intrinsic terminator for approximately 64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.

Mechanism of transcription termination: PTRF interacts with the largest subunit of RNA polymerase I and dissociates paused transcription complexes from yeast and mouse.

Transcription termination by RNA polymerase I (Pol I) is a stepwise process. First the elongating RNA polymerase is forced to pause by DNA-bound transcription termination factor (TTF-I). Then the ternary transcription complex is dissociated by PTRF, a novel factor that promotes release of both nascent transcripts and Pol I from the template. In this study we have investigated the ability of PTRF to liberate transcripts from ternary transcription complexes isolated from yeast and mouse. Using immobilized, tailed templates that contain terminator sequences from Saccharomyces cerevisiae and mouse, respectively, we demonstrate that PTRF promotes release of terminated transcripts, irrespective of whether mouse Pol I has interacted with the murine termination factor TTF-I or its yeast homolog Reb1p. In contrast, mouse Pol I paused by the lac repressor remains bound to the template both in the presence and absence of PTRF. We demonstrate that PTRF interacts with the largest subunit of murine Pol I, with TTF-I and Reb1p, but not the lac repressor. The results imply that Pol I transcription termination in yeast and mouse is mediated by conserved interactions between Pol I, Reb1p/TTF-I and PTRF.

A mutation in GRS1, a glycyl-tRNA synthetase, affects 3'-end formation in Saccharomyces cerevisiae.

3'-end formation is a complex and incompletely understood process involving both cis-acting and trans-acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a mutant hunt for strains defective in 3'-end formation was conducted. Following random mutagenesis, a temperature-sensitive strain exhibiting several phenotypes consistent with a role in transcription termination was isolated. First, readthrough of a terminator increases significantly in the mutant strain. Accordingly, RNA analysis indicates a decrease in the level of terminated transcripts, both in vivo and in vitro. Moreover, a plasmid stability assay in which high levels of readthrough lead to high levels of plasmid loss and transcription run-on analysis also demonstrate defective termination of transcription. Examination of polyadenylation and cleavage by the mutant strain indicates these processes are not affected. These results represent the first example of a transcription termination factor in Saccharomyces cerevisiae that affects transcription termination independent of 3'-end processing of mRNA. Complementation studies identified GRS1, an aminoacyl-tRNA synthetase, as the complementing gene. Sequence analysis of grs1-1 in the mutant strain revealed that nucleotides 1656 and 1657 were both C to T transitions, resulting in a single amino acid change of proline to phenylalanine. Further studies revealed GRS1 is essential, and the grs1-1 allele confers the temperature-sensitive growth defect associated with the mutant strain. Finally, we observed structures with some similarity to tRNA molecules within the 3'-end of various yeast genes. On the basis of our results, we suggest Grs1p is a transcription termination factor that may interact with the 3'-end of pre-mRNA to promote 3'-end formation.