JOURNAL OF BACTERIOLOGY, Nov. 2009, p. 7102–7108 0021-9193/09/$12.00 doi:10.1128/JB.00982-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 22
Archaeal Intrinsic Transcription Termination In Vivo䌤 Thomas J. Santangelo, L’ubomíra Cubonova´, Katherine M. Skinner, and John N. Reeve* Department of Microbiology, Ohio State University, Columbus, Ohio 43210 Received 25 July 2009/Accepted 31 August 2009
Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a -glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5ⴕ-TTTTTTTT (T8) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (tmcrA) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression ⬃90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region. Template DNAs were amplified from each T. kodakarensis strain, and transcription in vitro by T. kodakarensis RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5ⴕ-AAAAAAAA (A8) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination. for termination, the sites and efficiencies of termination in vitro were very sensitive to the assay conditions, changing with changes in the reaction temperature, nucleotide concentrations, salt solutions, or DNA template topology (36, 41, 43). Given these observations, validating in vivo studies of archaeal transcription termination are needed, but such studies have been precluded by the lack of a facile genetics. Fortunately, this barrier has been removed by the development of genetics for Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) (35, 38, 39), and we have taken advantage of this technology to place sequences predicted to be transcription terminators between a constitutive promoter and a reporter gene (34, 35), at a fixed site, in the T. kodakarensis genome (11). Based on quantitative effects on reporter gene expression, the results obtained confirm that an oligo(T) tract is sufficient to terminate archaeal transcription in vivo, and they establish that intrinsic archaeal termination is very different from intrinsic bacterial termination. The results are consistent with intrinsic archaeal termination resembling Pol III termination (36, 41), and they predict the existence of a mechanism that suppresses termination in vivo at oligo(T) sequences within genes.
Archaea are prokaryotes, with only one RNA polymerase (RNAP), genes cotranscribed in operons, and translation and transcription coupled (5, 10, 12). Archaeal RNAPs, however, are more similar to the three eukaryotic RNAPs (polymerases [Pol] I, II, and III) than to bacterial RNAPs (9, 19, 24, 25, 29, 48). In the absence of genetics, virtually all studies of archaeal transcription have used purified proteins and in vitro assays, and almost all have focused on initiation (13, 15, 29, 33, 36). Consistent with the Pol II-like structure of archaeal RNAPs, these studies have established that archaeal promoters have TATA boxes and transcription factor B recognition elements (BRE) and that initiation requires homologues of the eukaryotic TATA box binding protein (TBP) and transcription factor IIB (designated TFB in Archaea). Initiation is regulated by sequence-specific transcription factors that prevent or stimulate TBP, TFB, and/or RNAP binding to the promoter region (3, 13). In contrast to initiation, and despite the substantial differences between bacterial and eukaryotic transcription termination (4, 6, 8, 14, 16, 21, 27, 30, 49–51), very little research has addressed transcription termination in Archaea. The few in vitro studies reported (36, 41, 43) are consistent in concluding that termination is stimulated by oligo(T) sequences, but the extents of termination observed differed substantially, and identical oligo(T)-containing sequences positioned at different sites on the same template elicited different termination responses. Unlike intrinsic bacterial termination, termination was not limited to one or two discrete sites but often occurred at multiple adjacent sites, and not always within the oligo(T) sequence. Adding to the difficulty in defining the requirements
MATERIALS AND METHODS Construction of T. kodakarensis strains. T. kodakarensis TS372 was constructed previously from T. kodakarensis KW128 by transformation with plasmid pTS372 DNA (34). In the genome of T. kodakarensis TS372, homologous recombination upstream and downstream of TK1761 replaced the natural promoter and intergenic region upstream of this -glycosidase-encoding reporter gene with the strong constitutive archaeal promoter PhmtB and the intergenic sequence present upstream of TK1761 in pTS372 (34). Control experiments confirmed that TK1761 expression in T. kodakarensis TS372 is fully dependent on PhmtB and that the encoded -glycosidase is not essential and can be expressed at different levels without any detectable effects on growth (34, 35). All of the strains used in this study were constructed by the same plasmid transformation, homologous recombination, and selection procedures used previously to produce T. kodakarensis TS372 (33, 34, 39). The donor plasmid DNAs
* Corresponding author: Mailing address: Department of Microbiology, Ohio State University, Columbus, OH 43210. Phone: (614) 292-1267. Fax: (614) 292-8120. E-mail:
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were identical to pTS372 except for the additional presence of the experimental DNA sequence of interest (listed in Fig. 1) at position ⫹10 relative to the site of PhmtB-directed transcription initiation (⫹1) and 6 bp upstream of the ribosome binding site of TK1761 (see Fig. 2A). Each of these plasmids was constructed by cloning the sequence of interest into pTS372 (34) at the identical target site between PhmtB and TK1761. PCR amplification and sequencing of genomic DNA confirmed that the construct was integrated intact, at the desired location, in the genome of a representative transformant that was then used for further study. The T. kodakarensis strains constructed had only a single genomic copy of TK1761 and retained no additional plasmid-derived sequences. Aliquots of each plasmid DNA, and of the linear DNA molecules generated by PCR amplification from the genomic DNAs of transformants, were also used as templates for in vitro transcription assays. The primers used to generate templates for transcription added a 5⬘-biotinylated nucleotide to the template. Assay of TK1761 reporter gene expression. T. kodakarensis cultures were grown in ASW-YT medium (39), supplemented with 2 g of sulfur per liter, at 85°C to an optical density at 600 nm of ⬃0.6. The cells were harvested by centrifugation and lysed, and the -glycosidase activity present in the cleared lysate was measured by ortho-nitrophenyl--D-glucopyranoside (ONPG) hydrolysis, as previously described (34). Cleared lysates were obtained from at least three independent cultures of each strain, which had been grown on different days, and each lysate was assayed in triplicate. Synthesis of [32P]ApC, in vitro transcription, and separation and measurement of transcripts. The dinucleotide ApC (Sigma, St. Louis, MO) was incubated with T4 polynucleotide kinase and [␥-32P]ATP for 1 h at 37°C to synthesize [32P]ApC. The reaction mixture was then placed at 85°C for 10 min to inactivate the polynucleotide kinase. T. kodakarensis RNAP, TBP, and TFB2 (TK2287) were generated and purified as previously described (40). To allow open complex formation, an aliquot (10 nM) of the template DNA was incubated for 10 min at 85°C with 75 M [32P]ApC in a reaction mixture that contained 40 nM RNAP, 80 nM TFB2, 80 nM TBP, 20 mM Tris-HCl (pH 8), 250 mM KCl, 5 mM MgCl2, and 5 mM dithiothreitol. Transcription was initiated by adding a mixture of the four nucleoside triphosphates, each at a 1 mM concentration, and was allowed to proceed for 3 min at 85°C. The linear template DNA plus any attached paused or stalled transcripts were removed from an aliquot of each reaction mixture following transcription by binding and sedimentation on streptavidin-coated magnetic beads. Fully terminated transcripts were identified as transcripts that were released from the template and so remained in the supernatant. All of the [32P]-labeled transcripts synthesized in vitro were separated by electrophoresis through denaturing 20% (wt/vol) polyacrylamide gels, detected, and quantified by using a Storm 840 phosphorimager (GE Healthcare; Piscataway, NJ), as previously described (33, 36).
RESULTS An oligo(T) tract is sufficient to prevent downstream reporter gene expression. In in vitro assays, an oligo(T) sequence was sufficient to terminate archaeal transcription (36, 41, 43). T. kodakarensis TS552 was therefore constructed with 5⬘-TTT TTTTT (T8) positioned between the site of PhmtB-directed transcription initiation and TK1761 (Fig. 1A and 2A). The presence of T8 reduced the expression of the downstream TK1761 reporter gene by ⬃98% from that in T. kodakarensis TS372 (Fig. 1A). To investigate if an oligo(T) tract was necessary, or if an A/T-rich sequence was sufficient for this reduction in TK1761 expression, T. kodakarensis TS553 and TS554 were constructed with the 5⬘-AAAAAAAA (A8) sequence and the TA-repetitive sequence 5⬘-GCTAGTATATATA [designated (TA)4], respectively, positioned upstream of TK1761 (Fig. 1A). The presence of A8 had no inhibitory effect on TK1761 expression, but the presence of (TA)4 reduced -glycosidase activity by 64% relative to that in T. kodakarensis TS372 (Fig. 1A). Because the transition from archaeal transcription initiation to elongation is complete before position ⫹10 (36, 40), the sequences assayed for termination activity were positioned beginning at ⫹10. Nevertheless, to confirm that the inhibitory effect of T8 on TK1761 expression was not dependent on
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the ⫹10-to-⫹17 location, T. kodakarensis TS557 was constructed with an additional 20 bp preceding T8. The expression of TK1761 was reduced by ⬃97% in T. kodakarensis TS557, showing that T8 from ⫹30 to ⫹37 was just as effective in terminating transcription as T8 from ⫹10 to ⫹17 (Fig. 1A). As a control, T. kodakarensis TS437 was constructed with only the 20-bp sequence positioned upstream of TK1761, not followed by T8. The expression of TK1761 in T. kodakarensis TS437 was essentially the same as that in T. kodakarensis TS372 (data not shown). T. kodakarensis intergenic sequences inhibit downstream TK1761 expression. The GeSTer algorithm (45) predicts and ranks the likely efficiency of intrinsic bacterial terminators by their location and the presence of an inverted repeat followed by an oligo(T) (T-trail) sequence. This algorithm, with the inverted-repeat sequence requirement eliminated, was used to identify candidate transcription terminators within the T. kodakarensis genome. Twelve intergenic sequences so identified, which had one to three oligo(T) tracts ranging from T4 to T7, were PCR amplified and positioned upstream of TK1761 (Fig. 1B). In every strain, the presence of the T. kodakarensis intergenic sequence reduced TK1761 expression by ⬎70%, and in 8 of the 12 strains, the expression of TK1761 was reduced by ⬎90% (Fig. 1B). Mutations were introduced into several of these intergenic sequences to reduce any potential that the sequence upstream of the T tracts had to form a RNA hairpin. The mutated sequences were just as effective as the wild-type intergenic sequences in reducing TK1761 expression in vivo (data not shown). The inverted repeat in tmcrA is not required for intrinsic termination. Intrinsic bacterial terminators classically have an inverted-repeat sequence followed by an oligo(T) (T-trail) sequence (16, 27, 30, 37, 50), and mutations that reduce the ability of the inverted repeat to form a stable hairpin-loop dramatically reduce termination efficiency (32, 42, 46). Given this well-documented precedent, a sequence that conformed exactly to the bacterial terminator motif located immediately downstream of the very highly expressed mcrBDCGA operon in Methanothermobacter thermautotrophicus was predicted to be a transcription terminator. Northern blotting confirmed that termination in vivo occurred at or near this sequence, designated tmcrA (31). T. kodakarensis TS442 was constructed with tmcrA positioned upstream of TK1761, and this reduced TK1761 expression by ⬃88%, consistent with tmcrA functioning as an efficient transcription terminator in T. kodakarensis (Fig. 1C). To determine if the bacterial terminator motif was required for this activity, T. kodakarensis TS443, TS444, TS445, TS555, and TS556 were constructed with derivatives of tmcrA positioned upstream of TK1761 (Fig. 1C). The activity of tmcrA was not reduced by the presence of a point mutation in the inverted repeat (T. kodakarensis TS443) and was increased, rather than decreased, by the deletion either of parts of the inverted-repeat and loop region (T. kodakarensis TS444 and TS555) or of the entire region (T. kodakarensis TS556). In contrast, when the inverted-repeat and loop region was retained and the T-trail deleted (T. kodakarensis TS445), TK1761 expression increased relative to that in the strains containing an tmcrA-derived sequence that retained the T-trail (Fig. 1C). The -glycosidase activity in T. kodakarensis TS445 was nevertheless reduced to ⬃ 40% of that of T. kodakarensis
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FIG. 1. Genome organization, sequences assayed, and reporter gene expression in T. kodakarensis strains. (A) The trpE-TK1761 cassette, its transformation, and its recombination into the genome of T. kodakarensis KW128 to generate T. kodakarensis TS372 have been described in detail (34, 35). For each of the T. kodakarensis strains constructed, the synthetic oligonucleotide given adjacent to the TS strain designation was inserted, as indicated, beginning at position ⫹10 downstream of the site of PhmtB-directed transcription initiation (⫹1). The locations of the primers (sequences are available from T.J.S.) used to generate transcription templates from genomic DNAs are indicated by small filled rectangles. The -glycosidase activity encoded by the TK1761 gene present in each T. kodakarensis strain is shown in the bar graph as a percentage of the activity in T. kodakarensis TS372. The values are averages for triplicate assays of lysates from at least three independent cultures of each strain. (B) Sequences amplified from T. kodakarensis intergenic regions (IR) downstream from the genes listed (designated by “TK” followed by a number). Each sequence was positioned upstream of TK1761, as indicated in panel A, to generate the T. kodakarensis strain with the TS number given on the left. The -glycosidase activity present in each T. kodakarensis strain is shown in the bar graph, as in panel A, as a percentage of the activity in T. kodakarensis TS372. (C) The tmcrA sequence (31), positioned upstream of TK1761 in T. kodakarensis TS442, is shown with the 5⬘ and 3⬘ inverted-repeat sequences, intervening loop, and T-trail regions identified. The derivatives of tmcrA, and the mcrA coding sequences, which were identically positioned upstream of TK1761, are given to the right of the corresponding T. kodakarensis TS strain designations. The -glycosidase activity present in each T. kodakarensis strain is shown in the bar graph, as in panel A, as a percentage of the activity in T. kodakarensis TS372.
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FIG. 2. Template sequences and transcription termination in vitro. (A) Sequence of the PhmtB promoter and downstream region of the linear 372 template. This template was amplified, using primers that hybridized to regions (indicated by filled bars), from T. kodakarensis TS372 genomic DNA. The TATA box, site of transcription initiation ( ), ribosome binding site (RBS), and 5⬘ sequence of TK1761 (shaded box) are identified (34, 35). The additional sequences present in templates 552, 553, 554, and 557 are listed with the T8, A8, and (TA)4 sequences boxed. (B) Electrophoretic separation of the transcripts synthesized in vitro from linear (upper gel) and supercoiled, plasmid (lower gel) templates. The linear templates were generated by PCR as for panel A. The plasmid templates were the plasmids used to transform T. kodakarensis KW128 to generate T. kodakarensis strains TS372, TS552, TS553, TS554, and TS557. The [32P]ApC-labeled transcripts were separated by electrophoresis, visualized, and quantified using a Storm 840 phosphorimager (GE Healthcare). Transcripts that were terminated from ⫹10 on templates 372 and 557 are identified above and below the upper gel, respectively, by the nucleotides at that position in the 372 and 557 sequences (see panel A). The transcripts that were terminated within T8, A8, and (TA)4 on the linear templates are boxed. The regions of the gels containing abortive, terminated, runoff (2), or read-through ( ) transcripts are indicated. (C) Percentages of transcripts initiated and extended beyond ⫹10 that were terminated on linear versus circular versions of each template.
TS372, consistent with the T6 sequence in the loop region of tmcrA also having the capacity to decrease downstream gene expression. As controls, T. kodakarensis TS452 and TS454 were constructed, with M. thermautotrophicus coding sequences amplified from within mcrA, and similar in length to tmcrA, positioned between PhmtB and TK1761. The presence of these coding sequences reduced TK1761 expression by only ⬃25% and ⬃50%, respectively (Fig. 1C). Termination of transcription in vitro. To establish that the sequences inserted upstream of TK1761 did direct transcription termination in vitro, templates with a biotinylated 5⬘ nucleotide were PCR amplified from genomic DNA isolated from the T. kodakarensis strains. These templates were incubated in in vitro transcription reaction mixtures under condi-
tions in which every transcript synthesized was labeled equally, regardless of length, by the incorporation of [32P]ApC as the 5⬘ dinucleotide. Because all of the templates had the same promoter and sequence to ⫹10, as expected, initiation occurred with equal efficiency, and the patterns of abortive transcripts produced were very similar on all templates (Fig. 2B and 3A). Template removal by binding to streptavidin-coated beads removed ⬃25% of each runoff transcript, but all the other transcripts synthesized were fully terminated and released from the templates. The amount of [32P]ApC incorporated into all transcripts 10 nucleotides long or longer was measured, and this provided a value for the total number of transcripts initiated and extended beyond ⫹10. The percentage of this amount that was incorpo-
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rated into transcripts shorter than full-length transcripts provided a measure of termination on a particular template. Based on this calculation, ⬃35% of the transcripts initiated and extended beyond abortive transcripts on the control 372 template, amplified from T. kodakarensis TS372, were terminated before the RNAP reached the end of the template. Termination occurred most noticeably at positions from ⫹12 to ⫹20 but also at many other positions along the length of the 372 template (Fig. 2B). In contrast, on template 552, amplified from T. kodakarensis TS552, ⬎75% of the transcripts were terminated, and termination occurred predominantly at positions 4, 5, and 6 within the T8 sequence (Fig. 2B). Termination occurred to essentially the same extent (⬃82%) on template 557, amplified from T. kodakarensis TS557, again predominantly within the T8 sequence (Fig. 2B). As anticipated, based on the ⬃64% reduction of TK1761 expression in vivo by (TA)4 in T. kodakarensis TS554 (Fig. 1A), termination was ⬃20% higher on template 554 than on template 372 and occurred within the TA-repetitive sequence. In contrast, and in conflict with the lack of an inhibitory effect of A8 on TK1761 expression in T. kodakarensis TS553, ⬃75% of the transcripts initiated on template 553 were terminated, and substantial termination occurred at almost every position within the A8 sequence (Fig. 2B). To further investigate the A8 anomaly, in vitro transcription was repeated, but an aliquot of the supercoiled, circular plasmid DNA (pTS553) that had been used to transform T. kodakarensis KW128 to produce T. kodakarensis TS553 was used as the template. On this circular template, in addition to abortive and terminated transcripts, very long read-through transcripts were generated by continued transcription through terminators and around the circular DNA. Termination was calculated as the amount of [32P]ApC incorporated into terminated transcripts 10 nucleotides long or longer and was expressed as a percentage of the incorporation of [32P]ApC into all read-through and terminated transcripts. In contrast to the ⬃75% termination that occurred on the linear 553 template, only ⬃27% of the transcripts initiated on pTS553 DNA were terminated (Fig. 2B and C). Given this difference, transcription termination was also measured on pTS372, pTS552, pTS554, and pTS557, the plasmids used to generate T. kodakarensis TS372, TS552, TS554, and TS557 (Fig. 2B). There was a small (⬃10%) reduction in termination on pTS372 from that on the linear 372 template (Fig. 2C). The percentages of transcripts that terminated on pTS372 and pTS553 were essentially identical, consistent with the almost identical levels of -glycosidase activity present in T. kodakarensis TS372 and TS553 (Fig. 1A). In contrast to the topology-dependent termination at A8, topology had little effect on the extent of termination directed by T8 and (TA)4, but the predominant termination occurred at fewer adjacent sites in T8 and (TA)4 on the plasmid pTS552, pTS557, and pTS554 templates. We also investigated and compared transcription in vitro from both linear and plasmid templates that had the tmcrA and tmcrA-derived sequences (Fig. 3B). The extents of termination were essentially the same on the linear and circular templates, except for a small (⬃13%) increase in termination on pTS445 over that on the linear 445 template (Fig. 3C). The overall patterns of termination were, however, different. Consistent with the in vivo reporter assays, termination occurred on all
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templates at the T-trail sequence, with or without the presence of the inverted-repeat and loop region, but whereas this accounted for almost all of the termination on the plasmid templates (Fig. 3B), detectable termination also occurred at many other sites along the linear templates (Fig. 3A), including at the T6 sequence in the loop region of tmcrA. DISCUSSION All of the synthetic and natural oligo(T)-containing sequences positioned between PhmtB and TK1761 reduced the expression of the reporter gene in vivo, and the extents of reduction correlated well with the effectiveness of the sequence at terminating transcription in vitro by T. kodakarensis RNAP. It seems reasonable, therefore, to conclude that these sequences reduced TK1761 expression in vivo by directing intrinsic transcription termination upstream of TK1761. In contrast to intrinsic bacterial termination (9, 16, 27, 30, 37, 44, 50), this did not require the presence of an inverted repeat in the template sequence but was stimulated by the presence of an oligo(T) sequence, as is also the case for Pol III intrinsic termination (4, 26, 41). If the common ancestor of the archaeal RNAPs, Pol I, II, and III, was sensitive to oligo(T)-directed termination, the retention of this by Pol III may have been facilitated by the dedication of Pol III to the transcription of short RNA-encoding genes with few oligo(T) sequences. This argument cannot be extrapolated to all extant Archaea, since many have very A⫹T rich genomes, and although the T. kodakarensis genome is 52% G⫹C, it contains 7, 127, and 1,081 copies of T8, T7, and T6, respectively, of which 3, 80, and 713 are within rather than between genes. Given the demonstration of operon polarity (34), tight coupling of translation and transcription presumably suppresses intragenic transcription termination, but it seems likely that additional sequence information must inform a transcribing RNAP, either directly or via a transcription termination factor(s), to terminate or read through some oligo(T) sequences. T. kodakarensis grows optimally at 85°C, and such a hyperthermophilic lifestyle may preclude the use of nascent transcript folding to regulate gene expression during transcription. Given that this is a hyperthermophily limitation, it remains possible that spontaneous RNA duplex formation during transcription does direct termination in other Archaea, but inverted-repeat sequences occur only infrequently downstream of archaeal genes, arguing that this is not a common event (45, 47). To date, we have mapped the sites at which archaeal transcription is terminated in vitro on ⬎50 archaeal and bacterial intergenic sequences and synthetic templates, with a precision of 1 to 2 bp. With this database, we have sought to identify common sequence features and the driving force that destabilizes an otherwise very stable archaeal elongation complex (33, 36). Archaeal termination in vitro is stimulated by T-rich sequences of differing lengths and contexts, and it occurs most often within T tracts. However, as illustrated by the (TA)4 results (Fig. 2B), archaeal termination is not strictly dependent on an extended T tract, and in this regard, it may differ from Pol III termination. The predominance of termination at oligo(T) tracts is increased in vitro on circular versus linear templates (Fig. 2 and 3). In contrast to intrinsic bacterial termination, transcript release is not limited to one or two positions
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FIG. 3. Transcription termination in vitro by tmcrA on linear and circular templates. (A) Electrophoretic separation of the transcripts synthesized in vitro from linear templates amplified from T. kodakarensis TS372, TS442, TS443, TS444, TS445, TS555, and TS556. Templates 442, 443, and 445 have identical 5⬘ sequences (see Fig. 1C), and the transcripts that were terminated from ⫹10 on these templates are identified between the upper two gels by the nucleotide at that position. Transcripts that were terminated from ⫹ 10 on templates 555 and 556 are identified above and below the lower gel, respectively, by the nucleotide at that position. Transcripts that were terminated within the T-trail and loop regions of tmcrA are identified and boxed. (B) Electrophoretic separation of the transcripts synthesized from plasmid templates pTS372, pTS442, pTS443, pTS444, pTS445, pTS555, and pTS556. Transcripts that were terminated within T-trail sequences are boxed. The regions of the gels containing abortive, terminated, runoff (2) (A), or read-through ( , ) (B) transcripts are indicated. (C) Percentages of transcripts initiated and extended beyond ⫹10 that were terminated on linear versus circular versions of each template.
with defined spacing relative to a recognizable terminator motif but occurs at several adjacent sites, and not preferentially at sites where an RNA-DNA hybrid would have inherently weak base-pairing (7, 20, 23). With no requirement for an inverted repeat, there is no support for a mechanism dependent on a change in RNAP structure driven by RNA hairpin formation, and the data also provide no evidence for transcript slippage facilitating transcript release (1, 2, 9, 17, 28, 37, 44, 50, 51). Given this lack of support for any established mechanism, archaeal termination may be directed by a novel mechanism, but establishing this mechanism will require a combination of in vivo and in vitro assays. As the results with A8 exemplify, a sequence that terminates the transcription of a purified archaeal RNAP on a linear template in vitro may not terminate transcription in vivo. Similarly, in vivo validation is needed for the in vitro observation that archaeal transcription through an upstream sequence can influence an elongationtermination decision at a remote downstream sequence
(36). The occurrence of termination at multiple adjacent sites in vitro does, however, already have in vivo support. Based on high-resolution hybridization data, archaeal transcripts synthesized in vivo have a defined 5⬘ terminus but multiple 3⬘ termini (22). In Bacteria, many attenuators and riboswitches that regulate gene expression by controlling intrinsic termination have been identified (16, 18, 27, 30, 50). Intriguingly, to date, there is no experimental evidence or bioinformatics prediction for such regulation in Archaea, although long untranslated leader regions have been identified. Most analyses of archaeal genomes have likely searched primarily for sequences that conform to the bacterial intrinsic terminator motif, whereas the results reported here demonstrate that relatively simple oligo(T)-rich sequences could be sites at which regulation is imposed. The extent of intrinsic termination directed by the T-trail sequence of tmcrA, for example, could be reduced, as was observed in vivo under very high expression conditions (31), by the presence or
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absence of a factor interacting with the upstream inverted repeat.
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ACKNOWLEDGMENTS 27.
This research was supported by grants from the DOE (DE-FG0287ER13731) and NIH (GM53185), awarded to J.N.R., and by NIH fellowship (1F32-GM073336-01) support to T.J.S.
28.
REFERENCES
29.
1. Artsimovitch, I., and R. Landick. 1998. Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release. Genes Dev. 12:3110–3122. 2. Artsimovitch, I., and R. Landick. 2000. Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. Proc. Natl. Acad. Sci. USA 97:7090–7095. 3. Bartlett, M. S. 2005. Determinants of transcription initiation by archaeal RNA polymerase. Curr. Opin. Microbiol. 8:677–684. 4. Braglia, P., R. Percudani, and G. Dieci. 2005. Sequence context effects on oligo(dT) termination signal recognition by Saccharomyces cerevisiae RNA polymerase III. J. Biol. Chem. 280:19551–19562. 5. Cavicchioli, R. (ed.). 2007. Archaea. Molecular and cellular biology. ASM Press, Washington, DC. 6. Core, L. J., and J. T. Lis. 2008. Transcription regulation through promoterproximal pausing of RNA polymerase II. Science 319:1791–1792. 7. Datta, K., and P. H. von Hippel. 2008. Direct spectroscopic study of reconstituted transcription complexes reveals that intrinsic termination is driven primarily by thermodynamic destabilization of the nucleic acid framework. J. Biol. Chem. 283:3537–3549. 8. El Hage, A., M. Koper, J. Kufel, and D. Tollervey. 2008. Efficient termination of transcription by RNA polymerase I requires the 5⬘ exonuclease Rat1 in yeast. Genes Dev. 22:1069–1081. 9. Epshtein, V., C. J. Cardinale, A. E. Ruckenstein, S. Borukhov, and E. Nudler. 2007. An allosteric path to transcription termination. Mol. Cell 28:991–1001. 10. French, S. L., T. J. Santangelo, A. L. Beyer, and J. N. Reeve. 2007. Transcription and translation are coupled in Archaea. Mol. Biol. Evol. 24:893– 895. 11. Fukui, T., H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, and T. Imanaka. 2005. Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res. 15:352–363. 12. Garrett, R. A., and H.-P. Klenk. 2007. Archaea. Evolution, physiology, and molecular biology. Blackwell Publishing, Oxford, United Kingdom. 13. Geiduschek, E. P., and M. Ouhammouch. 2005. Archaeal transcription and its regulators. Mol. Microbiol. 56:1397–1407. 14. Gilmour, D. S., and R. Fan. 2008. Derailing the locomotive: transcription termination. J. Biol. Chem. 283:661–664. 15. Goede, B., S. Naji, O. von Kampen, K. Ilg, and M. Thomm. 2006. Proteinprotein interactions in the archaeal transcriptional machinery: binding studies of isolated RNA polymerase subunits and transcription factors. J. Biol. Chem. 281:30581–30592. 16. Grundy, F. J., and T. M. Henkin. 2006. From ribosome to riboswitch: control of gene expression in bacteria by RNA structural rearrangements. Crit. Rev. Biochem. Mol. Biol. 41:329–338. 17. Gusarov, I., and E. Nudler. 1999. The mechanism of intrinsic transcription termination. Mol. Cell 3:495–504. 18. Henkin, T. M. 2008. Riboswitch RNAs: using RNA to sense cellular metabolism. Genes Dev. 22:3383–3390. 19. Hirata, A., B. J. Klein, and K. S. Murakami. 2008. The X-ray crystal structure of RNA polymerase from Archaea. Nature 451:851–854. 20. Kashlev, M., and N. Komissarova. 2002. Transcription termination: primary intermediates and secondary adducts. J. Biol. Chem. 277:14501–14508. 21. Kawauchi, J., H. Mischo, P. Braglia, A. Rondon, and N. J. Proudfoot. 2008. Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination. Genes Dev. 22:1082–1092. 22. Koide, T., D. J. Reiss, J. C. Bare, W. L. Pang, M. T. Facciotti, A. K. Schmid, M. Pan, B. Marzolf, P. T. Van, F. Y. Lo, A. Pratap, E. W. Deutsch, A. Peterson, D. Martin, and N. S. Baliga. 2009. Prevalence of transcription promoters within archaeal operons and coding sequences. Mol. Syst. Biol. 5:285. 23. Komissarova, N., J. Becker, S. Solter, M. Kireeva, and M. Kashlev. 2002. Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell 10:1151– 1162. 24. Korkhin, Y., U. M. Unligil, O. Littlefield, P. J. Nelson, D. I. Stuart, P. B. Sigler, S. D. Bell, and N. G. Abrescia. 2009. Evolution of complex RNA polymerases: the complete archaeal RNA polymerase structure. PLoS Biol. 7:e102. 25. Kusser, A. G., M. G. Bertero, S. Naji, T. Becker, M. Thomm, R. Beckmann,
30. 31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45. 46. 47.
48. 49.
50. 51.
and P. Cramer. 2008. Structure of an archaeal RNA polymerase. J. Mol. Biol. 376:303–307. Landrieux, E., N. Alic, C. Ducrot, J. Acker, M. Riva, and C. Carles. 2006. A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation. EMBO J. 25:118–128. Merino, E., and C. Yanofsky. 2005. Transcription attenuation: a highly conserved regulatory strategy used by bacteria. Trends Genet. 21:260–264. Mooney, R. A., I. Artsimovitch, and R. Landick. 1998. Information processing by RNA polymerase: recognition of regulatory signals during RNA chain elongation. J. Bacteriol. 180:3265–3275. Naji, S., M. G. Bertero, P. Spitalny, P. Cramer, and M. Thomm. 2008. Structure-function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement. Nucleic Acids Res. 36:676–687. Nudler, E., and M. E. Gottesman. 2002. Transcription termination and anti-termination in E. coli. Genes Cells 7:755–768. Pihl, T. D., S. Sharma, and J. N. Reeve. 1994. Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum ⌬H. J. Bacteriol. 176: 6384–6391. Reynolds, R., R. M. Bermu ´dez-Cruz, and M. J. Chamberlin. 1992. Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. J. Mol. Biol. 224:31–51. Santangelo, T. J., L. Cubonova ´, C. L. James, and J. N. Reeve. 2007. TFB1 or TFB2 is sufficient for Thermococcus kodakaraensis viability and for basal transcription in vitro. J. Mol. Biol. 367:344–357. Santangelo, T. J., L. Cubonova ´, R. Matsumi, H. Atomi, T. Imanaka, and J. N. Reeve. 2008. Polarity in archaeal operon transcription in Thermococcus kodakaraensis. J. Bacteriol. 190:2244–2248. Santangelo, T. J., L. Cubonova ´, and J. N. Reeve. 2008. Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon. Appl. Environ. Microbiol. 74:3099–3104. Santangelo, T. J., and J. N. Reeve. 2006. Archaeal RNA polymerase is sensitive to intrinsic termination directed by transcribed and remote sequences. J. Mol. Biol. 355:196–210. Santangelo, T. J., and J. W. Roberts. 2004. Forward translocation is the natural pathway of RNA release at an intrinsic terminator. Mol. Cell 14: 117–126. Sato, T., T. Fukui, H. Atomi, and T. Imanaka. 2005. Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 71:3889–3899. Sato, T., T. Fukui, H. Atomi, and T. Imanaka. 2003. Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185:210–220. Spitalny, P., and M. Thomm. 2003. Analysis of the open region and of DNA-protein contacts of archaeal RNA polymerase transcription complexes during transition from initiation to elongation. J. Biol. Chem. 278:30497– 30505. Spitalny, P., and M. Thomm. 2008. A polymerase III-like reinitiation mechanism is operating in regulation of histone expression in Archaea. Mol. Microbiol. 67:958–970. Telesnitsky, A., and M. J. Chamberlin. 1989. Terminator-distal sequences determine the in vitro efficiency of the early terminators of bacteriophages T3 and T7. Biochemistry 28:5210–5218. Thomm, M., W. Hausner, and C. Hethke. 1994. Transcription factors and termination of transcription in Methanococcus. Syst. Appl. Microbiol. 16: 648–655. Toulokhonov, I., I. Artsimovitch, and R. Landick. 2001. Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins. Science 292:730–733. Unniraman, S., R. Prakash, and V. Nagaraja. 2002. Conserved economics of transcription termination in eubacteria. Nucleic Acids Res. 30:675–684. Uptain, S. M., C. M. Kane, and M. J. Chamberlin. 1997. Basic mechanisms of transcript elongation and its regulation. Annu. Rev. Biochem. 66:117–172. Washio, T., J. Sasayama, and M. Tomita. 1998. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination. Nucleic Acids Res. 26:5456–5463. Werner, F. 2008. Structural evolution of multisubunit RNA polymerases. Trends Microbiol. 16:247–250. West, S., N. J. Proudfoot, and M. J. Dye. 2008. Molecular dissection of mammalian RNA polymerase II transcriptional termination. Mol. Cell 29: 600–610. Yarnell, W. S., and J. W. Roberts. 1999. Mechanism of intrinsic transcription termination and antitermination. Science 284:611–615. Zhou, Y., D. M. Navaroli, M. S. Enuameh, and C. T. Martin. 2007. Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism. Proc. Natl. Acad. Sci. USA 104:10352– 10357.
