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Previous Article | Next Article 
Molecular and Cellular Biology, April 2006, p. 2688-2696, Vol. 26, No. 7
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.7.2688-2696.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532
Received 1 December 2005/ Returned for modification 23 December 2005/ Accepted 13 January 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most eukaryotic genes are transcribed by RNA polymerase II (Pol II) (23). Termination of transcription by Pol II is coupled to formation of the 3' end of the transcript (4, 35), which occurs by different mechanisms for protein-coding and noncoding RNAs. The 3' ends of mRNAs are formed by cotranscriptional endonucleolytic cleavage, followed by polyadenylation of the 3' end thus generated (49). The cis-acting elements that direct cleavage and polyadenylation in yeast are quite degenerate (22), but statistical analysis of a large number of known and putative processing sites has resulted in algorithms for the probabilistic prediction of mRNA 3' ends (20). Soon after transcript cleavage, termination of transcription and degradation of the 3' fragment of the cleaved transcript occurs, although the temporal order and interdependence of these two events are uncertain.
Formation of the 3' ends of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) differs between vertebrates and yeast. In vertebrates, most snoRNAs are processed from pre-mRNA introns (18), while snRNA genes utilize specialized promoters and 3'-end formation elements distinct from those that direct mRNA synthesis (24, 32). In contrast, all yeast snRNAs and most yeast snoRNAs are synthesized autonomously from promoters that appear similar to mRNA promoters. The 3' ends of these RNAs are generated by either endonucleolytic cleavage or termination, followed by trimming by the nuclear exosome, a complex of 3' exonucleases (6). In cases where endonucleolytic cleavage of snRNAs or snoRNAs is known to occur, it is not clear if this cleavage is coupled to termination.
We recently reported a mutational analysis of the terminator for a yeast snoRNA gene, SNR13 (39). The SNR13 terminator consists of at least two elements (regions I and II), which together span about 100 nucleotides. Here we place the SNR13 terminator in the 3' untranslated region (UTR) of a protein-coding gene and find that, surprisingly, it directs polyadenylation of the mRNA. The polyadenylation sites map immediately downstream of region II, which contains sequences similar to mRNA cleavage and polyadenylation elements. A parallel analysis of the SNR65 snoRNA terminator yields similar results, suggesting that bipartite terminators containing cleavage and polyadenylation signals may be a common characteristic of yeast snoRNA genes. Using both the SNR13 snoRNA terminator and the CYC1 mRNA terminator, we selected for trans-acting mutations that result in terminator readthrough and obtained substitutions in the Rpb3 and Rpb11 subunits of Pol II. These subunits form a heterodimer that is homologous to the 
subunit homodimer of bacterial RNA polymerase and is stabilized by a long coiled-coil interaction. The readthrough mutations cluster at the extreme C terminus of the Rpb11 dimerization helix and an interacting residue in Rpb3. We propose that this surface is a contact point for factors that transmit the termination signal to Pol II.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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UTR lacks the CUP1 3' UTR but retains the PGK1 3' UTR and was created by replacing a 751-bp BamHI-SalI fragment of pGAC24 with a 594-bp BamHI-SalI fragment of a PCR product made with the 5'-CUP and CUP1-3'-SalI primers. The 217-bp SNR65/RPS14A intergenic region was amplified by PCR with flanking XhoI sites with primers SNR65-XHO-101F and SNR65-XHO-317R and inserted into the intronic XhoI site of pGAC24 to create pGAC24-SNR65F and pGAC24-SNR65R or into the SalI site of pGAC24-UTR to create pGAC24-65UTR-F and pGAC24-65UTR-R. A single-nucleotide substitution of C for T is encoded within the SNR65-XHO-101F primer relative to the sequence of the SNR65/RPS14A intergenic region currently deposited in the Saccharomyces Genome Database (www.yeastgenome.org/) due to an update in the database occurring after the primers were created. Mutational analysis and mapping of polyadenylation sites were carried out with constructs having this variant sequence. A primer with a sequence matching the current database entry, SNR65-XHO-101F-T, was subsequently used to create versions of pGAC24-SNR65F and pGAC24-SNR65R that perfectly match the SGD sequence. No difference in copper sensitivity was observed with these corrected constructs compared to the variants. The corrected SNR65-XHO101F-T primer was used with SNR65-XHO-236R to create pGAC24-SNR65-101-236. RNA analysis. Northern blots were prepared as described previously (39), with 20 µg of total cellular RNA per lane, and probed with 32P-labeled oligonucleotide 3'CUP-2 or ACT1-probe. To map sites of polyadenylation, 0.1 to 1 µg of total cellular RNA prepared by the glass bead-guanidinium isothiocyanate-hot phenol method (45) was combined with 250 pmol of T16-BSG1 primer in 26.5 µl of H2O, incubated at 95°C for 10 min and 65°C for 10 min, and cooled on ice. Reaction mixtures were brought to 50 µl by the addition of 5 µl of 100 mM dithiothreitol, 5 µl of deoxynucleoside triphosphates (10 mM each), 1 µl of RNasin (Promega), 10 µl of 5x avian myeloblastosis virus reverse transcriptase buffer, and 2.5 µl of avian myeloblastosis virus reverse transcriptase (USB) and incubated at 42°C for 1 h. One-fifth of the reverse transcription (RT) reaction mixture was used directly in PCRs with the T16Bsg1 and 5'-CUP primers and Tfl DNA polymerase (Epicenter Technologies). Products were digested with BamHI and SalI, cloned into pUC118, and sequenced with M13 universal primers.
Isolation of cis-acting readthrough mutations. Error-prone PCR of the ACT-CUP intron region of pGAC24-SNR65F, cotransformation of PCR products with gapped pGAC24, and selection of copper-resistant colonies were carried out as described previously (39).
Isolation of trans-acting readthrough mutations.
The genome-wide selection for spontaneous SNR13 terminator readthrough mutations was described previously (39). One mutant (from the 46a background; MATa cup1 ura3 his3 trp1 lys2 ade2 leu2) having a cold-sensitive growth phenotype was transformed with a YCp50-based yeast genomic library to identify clones that enable growth at 16°C. A single complementing clone with a genomic insert of 
14.4 kb containing RPB11 and several other genes was identified, and subcloning revealed that the complementing activity was contained within a 1.55-kb EcoRI-SacI fragment containing only RPB11 and the extreme 5' end of SIN3. Sequencing of the genomic RPB11 locus in the mutant strain revealed a GAG-to-GGG mutation in codon 108.
DNA fragments containing wild-type RPB3, RPB7, and RPB11 alleles were amplified from yeast strain 46 (isogenic with 46a except MAT) with KOD Hi-Fi DNA polymerase (Novagen) and cloned into URA3-marked centromere plasmid pRS316. Mutagenesis was carried out by PCR of these plasmid clones with Tfl DNA polymerase (Epicenter) with universal M13 forward and reverse primers and deoxynucleoside triphosphates at 200 µM each. The resultant PCR products were cotransformed with EcoRI/BamHI-digested pRS316 plasmid DNA into yeast strain 46 harboring LEU2-marked reporter plasmid pGAC24-SNR13 (40) or pGAC24-CYC83F (39). Transformants were selected on medium lacking uracil and leucine, and colonies were then replica plated onto medium lacking leucine and containing 0.15 or 0.25 mM CuSO4 to select for terminator readthrough. To test for linkage of the copper-resistant growth phenotype to the mutagenized plasmid, strains were streaked onto plates containing 5-fluoroorotic acid and lacking leucine to select for loss of the URA3-marked plasmid and then scored for loss of copper resistance.
Plasmid-borne Pol II subunit genes from copper-resistant strains were amplified with KOD Hi-Fi DNA polymerase with M13 universal primers and sequenced with a combination of M13 and gene-specific primers. PCR products from selected RPB11 mutants (see Table 2) were cloned into TRP1-marked centromere plasmid pRS314 to confirm linkage of the copper resistance phenotype and to test for function in the absence of wild-type RPB11. The genomic RPB11 locus of 46 bearing a second copy of RPB11 on URA3-marked centromere plasmid pRS316 was disrupted by transformation with an rpb11::KanMX4 fragment amplified from an rpb11 disruption strain obtained from Research Genetics and selection for G418 resistance. Integration at the RPB11 locus was confirmed by PCR and by dependence on a plasmid-borne RPB11 allele for viability. Mutant rpb11 alleles in pRS314 were transformed into 46 rpb11, tested for terminator readthrough activity by plating on medium containing copper, and tested for function in the absence of RPB11 by plating on medium containing 5-fluoroorotic acid. All of the mutant alleles tested (see Table 2) conferred resistance to at least 0.15 mM copper.
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RESULTS |
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50-bp sequences designated region I and region II. Region I contains sequences expected to bind Nrd1, while region II contains features similar to pre-mRNA cleavage-polyadenylation sites. Each region has weak terminator activity, but both are required for efficient termination and point mutations in both are required for strong readthrough (39).
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It was anticipated that insertion of the 217-bp SNR65/RPS14A intergenic fragment into the ACT/CUP intron in either orientation would result in decreased expression of the ACT/CUP gene, due to either the SNR65 terminator in the forward orientation or the RPS14A cleavage-and-polyadenylation site in the reverse orientation. Surprisingly, while the forward orientation conferred severe copper sensitivity (SNR65long, Fig. 1C), the reverse orientation did not (data not shown). Thus, the RPS14A cleavage-and-polyadenylation site does not appear to function in the context of the ACT/CUP intron.
We examined the role of the Nrd1 protein in SNR65 terminator function by testing copper sensitivity in a nrd1-5 mutant strain (Fig. 1C). No appreciable increase in copper resistance was imparted by the nrd1-5 mutation with the SNR65long insertion, suggesting that Nrd1 may not be required for SNR65 terminator function. However, sequence alignment of the SNR65 3'-flanking region revealed two blocks of similarity with the SNR13 terminator (Fig. 2), corresponding roughly to regions I and II but separated by a spacer region of about 50 nucleotides. Furthermore, region I of SNR65 is highly similar to a region of the NRD1 5' UTR that is required for negative autoregulation of Nrd1 level by premature transcription termination (40) (Fig. 2). Therefore, we tested whether a smaller segment of SNR65 3'-flanking sequence containing the region I-like and spacer sequences but not region II (nucleotides 101 to 236) directs Nrd1-dependent termination. Indeed, this smaller region (SNR65short) causes moderate copper sensitivity that is relieved by the nrd1-5 mutation (Fig. 1C).
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To identify nucleotides critical for SNR65 terminator function, we generated a pool of mutated DNA fragments containing the entire SNR65/RPS14A intergenic region by error-prone PCR and introduced them into the ACT/CUP fusion gene intron in the forward orientation by in vivo homologous recombination. Yeast transformants that survived on plates containing 0.15 mM copper, a concentration restrictive for the wild-type insert, were selected. The ACT/CUP plasmid was rescued from 17 copper-resistant transformants, plasmid linkage of copper resistance was confirmed by retransformation, and the insert from each plasmid was sequenced. The results are displayed in Table 1 and Fig. 2. Interestingly, 14 of the 17 plasmids have mutations affecting the UA dinucleotide at positions 260 and 261, and all 17 have at least one mutation in region II. Mutation of A251, U260, or A261 alone is sufficient to allow growth on 0.15 mM copper. Growth on 0.7 mM copper was observed only when additional mutations are present, either another mutation in region II (A283G) or several mutations in region I (U166A, C172A) and the spacer (U198). The failure to obtain mutations in region I alone suggests that region II is the major determinant for termination on SNR65 and/or that region I of SNR65 has redundant elements not easily disrupted by random mutagenesis.
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strain, indicating that functional ACT/CUP mRNA production was not significantly impaired. However, the SNR13 forward insert did result in slower growth on 0.7 to 1.0 mM copper plates, which could be rescued by a mutation in the gene for Nrd1 (data not shown). Total cellular RNA from the transformants was analyzed by Northern blotting with a probe complementary to CUP1 (Fig. 3B). In the reverse orientation, the SNR13 insert increases the length of the ACT/CUP mRNA but apparently does not result in any new 3' ends (Fig. 3B, compare lanes 1 and 3). This result was expected since the reverse orientation should encode no 3'-end formation elements. There does appear to be a decrease in the steady-state level of the mRNA, perhaps because the altered 3' UTR sequence destabilizes the transcript.
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The SNR65 terminator also elicited polyadenylation in the ACT/CUP 3' UTR. The 217-bp fragment bearing the SNR65 terminator contains the RPS14A cleavage-and-polyadenylation site in the opposite orientation, so it was expected to direct 3'-end formation in either orientation. Indeed, both the forward and reverse orientations of the insert direct the formation of shorter transcripts than are seen in the absence of an insert (Fig. 3B, compare lanes 4 and 5 with lane 6), indicative of cleavage and/or termination within the insert. Both the SNR65 and RPS14A terminator elements appear to be more efficient than the SNR13 terminator in the context of the ACT/CUP 3' UTR, as little or no utilization of the PGK1 cleavage-and-polyadenylation site is apparent. The fact that the transcripts produced from the reverse orientation of the SNR65 insert are approximately the same length as those produced from the forward orientation of the SNR13 insert (Fig. 3B, compare lanes 2 and 4) indicates that the RPS14A polyadenylation site is about 100 nucleotides downstream of the stop codon, between regions I and II of the SNR65 terminator (Fig. 1A). Indeed, of seven cDNAs analyzed for the reverse SNR65 insert, six have poly(A) sites between +205 and +220 (Fig. 2, in the opposite strand of that shown). The seventh clone has a poly(A) site at position +181. Thus, the SNR65 and RPS14A terminators are indeed interdigitated on opposite strands.
The polyadenylation sites for the SNR65 terminator were mapped by the same method as that described for the SNR13 terminator, and the results are displayed in Fig. 2. Eight cDNA clones were analyzed; four had poly(A) sites at position +303, and the remainder had sites between +287 and +297. Thus, as for SNR13, the SNR65 poly(A) sites are at the downstream end of region II. It therefore appears that at least two snoRNA terminators contain cleavage and polyadenylation signals at their downstream ends, although we cannot exclude the possibility that the poly(A) tail derives from termination followed by cleavage-independent polyadenylation by Trf4 (see Discussion).
trans-acting terminator readthrough mutations in Pol II subunits. We have also used the ACT-CUP reporter to select for mutations in trans-acting factors that induce readthrough of the SNR13 terminator (38-40). The factors thus identified include two RNA-binding proteins, Nrd1 and Nab3, a helicase, Sen1, and a protein phosphatase, Ssu72. Nrd1 and Sen1 bind to the C-terminal domain of the largest subunit of Pol II, Rpb1 (42, 48), while Ssu72 binds to the second largest Pol II subunit, Rpb2 (16), and utilizes the Pol II C-terminal domain as a substrate (29). Nab3 binds to Nrd1 (10). A plausible model for the function of these four factors is that Nrd1 and Nab3 recognize terminator sequences in the nascent transcript, possibly assisted by Sen1, and transmit the termination signal to Pol II, perhaps via Ssu72. The mechanism by which Pol II receives the termination signal is unknown, and no mutations in Pol II subunits had been obtained in the readthrough selection.
Here we report the identification of terminator readthrough mutations in two subunits of Pol II. Selection for spontaneous copper-resistant mutants with a haploid cup1 yeast strain bearing the ACT/CUP fusion gene with the SNR13 terminator inserted in the intron yielded a cold-sensitive mutation that was complemented by a plasmid bearing RPB11, the gene encoding the second smallest subunit of Pol II (46). The RPB11 locus was amplified from the cold-sensitive strain by PCR and sequenced, revealing a missense mutation that substitutes glycine for glutamate at residue 108 of the 120-amino-acid Rpb11 protein (rpb11-E108G). When cloned into a low-copy-number plasmid and transformed into a wild-type RPB11 strain, the rpb11-E108G allele dominantly confers readthrough of the SNR13 terminator (data not shown), as well as the poly(A) site/terminator from the protein-coding CYC1 gene (Fig. 4A). These results confirm that the rpb11-E108G mutation is solely responsible for the copper-resistant phenotype and demonstrate that the substitution in Rpb11 affects recognition of both a snoRNA and an mRNA terminator.
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-helix that forms a coiled-coil interaction with the third largest subunit of Pol II, Rpb3 (12). To search for other substitutions in the Rpb3/11 heterodimer that interfere with termination, RPB3 and RPB11 were randomly mutated by error-prone PCR and introduced into the reporter strain by transformation. Transformants were selected for dominant induction of readthrough of either the SNR13 or the CYC1 terminator. As a control, randomly mutated RPB7 was put through the same selection. The Rpb7 subunit maps to a different face of Pol II than the Rpb3/11 heterodimer (2, 5) (Fig. 5). Very few copper-resistant colonies were obtained after transformation with mutated RPB7, and these strains retained copper resistance after selection against the URA3-marked RPB7 plasmid, indicating that readthrough was due to spontaneous mutations in the genome or ACT/CUP reporter. In contrast, hundreds of copper-resistant colonies were obtained after transformation with mutated RPB11. When 15 of these candidate RPB11 mutant strains were subjected to selection against the URA3-marked plasmid, all 15 lost copper resistance, indicating that RPB11 plasmid-linked mutations could readily be selected. Copper-resistant strains were also readily obtained with mutated RPB3. The RPB3 mutant candidates were not subjected to the plasmid linkage test, but sequencing of the plasmid-borne RPB3 gene from 12 candidates revealed that they all contain the same substitution (see below).
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Strikingly, all other mutations selected in RPB11 result in changes C terminal to E108 (Fig. 4B). The most common of these were the substitutions L111P and L114P. In addition, nonsense mutations were obtained at codons 109, 112, and 114, either alone or in combination with a substitution (Table 2). Thus, alterations in the structure of the extreme C-terminal segment of Rpb11 appear to result in inefficient recognition of terminators by Pol II. The fact that the readthrough mutations are dominant indicates that the alterations in Rpb11 do not prevent assembly into Pol II, nor do they strongly inhibit initiation or elongation by Pol II.
All 12 Rpb3 mutants identified in the selection encode a substitution of glutamate for lysine 9 (K9E, Fig. 4C). Remarkably, K9 of Rpb3 lies within a few angstroms of Rpb11-E108 in crystal structures of yeast Pol II (Fig. 5). Rpb3-K9 is not part of the C-terminal -helix that forms a coiled coil with Rpb11; rather, it is in a ß-sheet that cradles the C terminus of the Rpb11 -helix. The asymmetry of the selected substitutions in Rpb11 and Rpb3 suggests that it is not simply disruption of the coiled coil that results in readthrough, but rather alterations in the surface at one end of the coiled coil. A different domain of Rpb3 was previously implicated in the activation of transcription (41); our mutations are the first evidence for a function of the Rpb3/11 heterodimer in termination.
The basic side chain of Rpb3-K9 and the acidic side chain of Rpb11-E108 have the potential of forming an ionic interaction. However, this potential is not conserved in several of the known orthologs of yeast Rpb3/11 (Fig. 4). Furthermore, the recessive lethal growth defect of an rpb11-E108K mutant strain is not rescued by transformation with rpb3-K9E on a low-copy-number plasmid (data not shown), suggesting that an ionic interaction between these two residues is not sufficient for normal Rpb3/11 activity.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SNR13 and SNR65 terminators contain polyadenylation signals. The terminators from two yeast snoRNA genes, SNR13 (7, 33, 39) and SNR47 (7), have previously been studied in some detail. Both terminators are bipartite in structure, with functional elements centered roughly 45 bp apart. We have now characterized a third snoRNA terminator, that of SNR65. Interestingly, it too is bipartite, but the region I and region II elements are centered about 95 bp apart, due to the presence of a 50-bp spacer sequence. Within this spacer lies the cleavage-and-polyadenylation site for the convergently transcribed gene RPS14A. Thus, separation of regions I and II may be an adaptation to allow interdigitated terminators on opposite strands. Such flexibility in terminator architecture contributes to the difficulty in identifying terminators by sequence alone. Interestingly, regions I of the SNR13 and SNR65 terminators, and the NRD1 autoregulatory element, share fairly extensive sequence similarity (Fig. 2), which suggests that natural terminators are more than just a collection of low-affinity Nrd1 (GUAA) and Nab3 (UCUU) binding sites (7).
We previously noted that region II of the SNR13 terminator has sequence similarity to yeast cleavage-and-polyadenylation sites (39). Here we show that both the SNR13 and SNR65 terminators direct polyadenylation when placed in the 3' UTR of a reporter gene. In either case, polyadenylation occurs at the downstream boundary of region II, within a few nucleotides of sites predicted by the algorithm of Graber et al. (20), which was developed to identify cleavage-and-polyadenylation sites in protein-coding genes. We therefore presume that these polyadenylated 3' ends are produced by the cleavage and polyadenylation factors that act on mRNAs. While we cannot rule out the possibility that these 3' ends are generated by termination and are subsequently polyadenylated by Trf4, which marks nuclear RNAs for exosomal degradation (25, 30, 43, 47), we consider this scenario unlikely since the transcripts accumulate to high levels and are translated efficiently, judging by their ability to confer copper resistance. Furthermore, our observations are consistent with the fact that several cleavage and polyadenylation factors have been implicated in termination on snoRNA genes. These include CFIA subunits Rna14 and Rna15 (17, 33) and holo-CPF subunits Pti1, Ref2, Ssu72, and Swd2 (9, 14-16, 19, 34, 39).
Earlier studies detected polyadenylated forms of two noncoding RNAs, telomerase RNA (8) and U2 snRNA (1). Approximately 5 to 10% of telomerase RNA is polyadenylated in wild-type yeast cells, whereas polyadenylation of U2 RNA is observed only when processing of precursor U2 RNA by Rnt1 endonuclease is inhibited. Several snoRNA genes produce extended and polyadenylated transcripts when the nuclear exosome is inhibited by deletion of the gene for the Rrp6 subunit, and this polyadenylation is dependent on Pap1, the poly(A) polymerase that acts on mRNAs (44). It is not clear if the polyadenylation sites of these noncoding RNAs map to the 3' ends of their terminator sequences and thus are mechanistically similar to cleavage and polyadenylation directed by the SNR13 and SNR65 terminators.
It is intriguing that the SNR65-RPS14A intergenic region did not confer copper sensitivity when inserted into the ACT/CUP fusion gene intron in the RPS14A orientation, yet it efficiently directed cleavage and polyadenylation when inserted into the ACT/CUP 3' UTR. One interpretation of this result is that the RPS14A cleavage-and -polyadenylation signal can only be recognized after Pol II has transcribed several hundred nucleotides of RNA. In contrast, the CYC1 cleavage-and-polyadenylation signal strongly decreases ACT/CUP gene expression when inserted into the intron (Fig. 4A). The CYC1 coding region is only 330 bp long, while the RPS14A coding region (with intron) is 721 bp long. Thus, the dichotomous behavior of the terminator elements of these two genes may reflect their adaptation to different gene lengths. This model is consistent with the finding that Nrd1, Nab3 (34), and Sen1 (E.J.S. and D.A.B., unpublished data) exhibit high occupancy over both the 5' and 3' ends of tested protein-coding regions, suggestive of recruitment upon initiation of transcription, while cleavage and polyadenylation factors peak primarily over the 3' end of coding regions, implying recruitment only after significant elongation (28, 34). We propose that the Nrd1-dependent termination pathway is most active within a few hundred base pairs of the transcription start site, while the cleavage-and-polyadenylation-dependent termination pathway operates primarily at sites several hundred base pairs downstream of the start site. Intermediate sites, such as the CYC1 poly(A) site, might utilize components of both pathways.
Substitutions in the Rpb3/11 heterodimer cause terminator readthrough. How might the selected substitutions in the Rpb3/11 heterodimer induce terminator readthrough? The location of the substitutions on the rear surface of Pol II suggests that they may interfere with the interaction of Pol II with an extrinsic termination factor. Termination of Pol II transcription appears to require the binding of protein factors to terminator elements in the nascent transcript (3, 37). Although the Rpb3/11 mutations are distant from the RNA exit pore (Fig. 5), a basic channel that may provide a path for the nascent transcript ("groove 2" of reference 11) leads from the RNA exit pore to the vicinity of Rpb3/11. Therefore, a termination factor that binds to the nascent transcript may contact Pol II at the Rpb3/11 heterodimer. Such a factor may induce termination by causing an allosteric change in the elongating polymerase, or it may reach around the back of the enzyme and interact more directly with macromolecules in or near the active site. It is interesting that the Rpb3/11 heterodimer plays a central role in the interaction of Pol II with the 21-subunit Mediator protein complex (13). While Mediator functions in initiation of transcription and its involvement in termination has not been proposed, one can imagine that binding of Mediator to Pol II at the end of a gene may serve to couple termination to reinitiation.
There are several other possible candidates for a factor that interacts with Rpb3/11 to promote termination. Because our mutations cause readthrough of both a snoRNA and an mRNA terminator, factors known to be common to both pathways are of particular interest. Such factors currently include the cleavage and polyadenylation factors mentioned above, as well as the helicase Sen1 (38, 40; E.J.S. and D.A.B., unpublished). Given that the Rpb3/11 heterodimer is homologous to the N-terminal domains of the subunit homodimer in bacterial RNA polymerase, it is intriguing that mutations in the Escherichia coli subunit C-terminal domain have been shown to cause readthrough of terminators that are dependent on the helicase rho (26). These observations raise the possibility that the signaling pathway for transcription termination in eukaryotes and prokaryotes may employ analogous interactions between a transcript-associated helicase and the upstream end of RNA polymerase.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant GM44665.
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FOOTNOTES |
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