When Rnap Transcribes 2-9 Nt
J Biol Chem. 2011 Sep 9; 286(36): 31576–31585.
RNA Folding in Transcription Elongation Complex
IMPLICATION FOR TRANSCRIPTION TERMINATION* 
Lucyna Lubkowska
From the ‡NCI Heart for Cancer Research, Frederick Cancer Inquiry and Development Center, Frederick, Maryland 21702,
Anu Southward. Maharjan
the §Program in Genomics and Differentiation, NICHD, National Institutes of Health, Bethesda, Maryland 20892,
¶Smith Higher, Northampton, Massachusetts 01063, and
the ‖Current Graduate Program, Rice University, Houston, Texas 77005
Natalia Komissarova
From the ‡NCI Center for Cancer Inquiry, Frederick Cancer Research and Development Centre, Frederick, Maryland 21702,
the §Plan in Genomics and Differentiation, NICHD, National Institutes of Wellness, Bethesda, Maryland 20892,
the ‖Current Graduate Plan, Rice University, Houston, Texas 77005
Received 2011 April 8; Revised 2011 Jun 20
- Supplementary Materials
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Supplemental Data
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Abstract
Intrinsic transcription termination signal in DNA consists of a brusk inverted repeat followed past a T-rich stretch. Transcription of this sequence by RNA polymerase (RNAP) results in formation of a "termination hairpin" (TH) in the nascent RNA and in rapid dissociation of the transcription elongation complex (EC) at termination points located 7–8 nt downstream of the base of TH stalk. RNAP envelops 15 nt of the RNA following RNA growing 3′-terminate, suggesting that folding of the Thursday is impeded past a tight protein surroundings when RNAP reaches the termination points. To monitor TH folding under this constraint, nosotros halted Escherichia coli ECs at diverse distances downstream from a TH and treated them with unmarried-strand specific RNase T1. The EC interfered with TH germination when halted at 6, 7, and eight, but not ix, nt downstream from the base of the potential stem. Thus, immediately before termination, the downstream arm of the TH is protected from complementary interactions with the upstream arm. This protection makes Th folding extremely sensitive to the sequence context, considering the upstream arm easily engages in competing interactions with the rest of the nascent RNA. We demonstrate that past de-synchronizing Thursday germination and transcription of the termination points, this subtle contest significantly affects the efficiency of transcription termination. This finding tin explain previous puzzling observations that sequences far upstream of the TH or betoken mutations in the terminator that preserve Thursday stability impact termination. These results can assistance understand other time sensitive co-transcriptional processes in pro- and eukaryotes.
Keywords: Bacteria, RNA Folding, RNA Polymerase, RNA Structure, Transcription Termination
Introduction
RNA is involved in nearly every aspect of factor expression, and the germination of specific three-dimensional structures is ofttimes crucial for RNA functionality. Amongst other biological processes, RNA conformation is important for translation (1, 2), plasmid replication (3, iv), RNA splicing (5), RNAi-induced factor silencing (6–eight), gene command by riboswitches (9), transcription pausing, antitermination, and termination (10–xiv).
In the jail cell, folding of RNA occurs co-transcriptionally. This fact dictates the folding pathway because the upstream part of RNA can fold before the downstream part is synthesized by RNA polymerase (RNAP),iii (xv–17). Such sequential folding leads to aggregating of metastable folding intermediates, which can forbid or delay refolding of the RNA into the thermodynamically favorable and functional conformation upon completion of the transcript synthesis. Such kinetic traps can be affected by variation in the rate of RNA elongation. For example, transcription of bacterial ribosomal RNA genes, or of a replication primer for ColE1 plasmid, by a foreign RNAP from phage T7 results in germination of non-functional RNAs, presumably because T7 RNAP is faster than the cognate Escherichia coli RNAP (18, 19). A delay in RNA folding into functionally agile conformation can give a fourth dimension window for regulation of fourth dimension sensitive biological processes, in which RNA structure is involved. These processes include translation (20), ribosome associates (21, 22), and culling splicing (5, 23, 24). Time-sensitive processes that are tightly coupled with transcription, including RNA splicing, transcription pausing, termination, and antitermination are expected to be peculiarly sensitive to the kinetic traps.
To understand the effect of RNA folding on the co-transcriptional processes, one should keep in mind that folding occurs in the context of the elongation circuitous (EC), the highly stable ternary complex formed by RNAP, DNA, and nascent RNA. The stable EC falls apart during termination. In prokaryotes, intrinsic termination is triggered by a brusk stalk-loop structure formed by the nascent RNA ("termination hairpin", TH) followed past a run of U residues (Fig. i A and Ref. 25). RNA synthesis stops and the EC dissociates at the termination points, typically located at the 7th and 8th U of the run (henceforward chosen the h+7 and h+8 positions, respectively, Fig. 1 A).
The termination hairpin and the structure of transcription elongation complex. A, Tr2 terminator of E. coli bacteriophage lambda used as a model in our study shown schematically in the context of the EC. The RNA is represented by red line and messages, the Deoxyribonucleic acid, past light grey lines, which separate to evidence 12 nt transcription bubble partially occupied by an viii nt RNA:Dna hybrid. RNAP is shown past a gray oval. B, construction of the EC from Ref. 28. DNA (night grey) and RNA (red) are shown in CPK representation. The β-subunit and β′ subunit domain between amino acids 380–410 (medium gray) are shown in surface representation. β′ structural features (imperial) contacting RNA in the exit channel are shown in tube representation. The rest of the β′ subunit, which obscured the view of the RNA exit channel, was removed from the construction to demonstrate that fifteen iii′-terminal RNA residues are sequestered in the enzyme. Numbers indicate DNA and RNA nt covered by RNAP and the length of RNA:DNA hybrid.
In elongation, RNAP (consisting of v subunits, α2β′βω) forms a clench embracing the DNA and the RNA (Fig. 1 B). The 3′-cease of the RNA is kept at the catalytic center of the enzyme positioned on the bottom of the clamp (26–28). The two Deoxyribonucleic acid strands are separated in the region of the hybrid, forming transcription bubble (29). RNAP covers 20 nt of the DNA downstream and fifteen nt upstream from the catalytic center (26). Eight 3′-proximal nt of the RNA are base-paired to the template Deoxyribonucleic acid strand forming RNA:DNA hybrid (30, 31). Across the 8 nt, the RNA is segregated from the template into a narrow cylindrical channel on the interface of the two largest subunits, β′ and β (28). This RNA go out aqueduct encloses 7 bases of the unmarried-stranded RNA (28, 32). Simply RNA residues located farther than 15 nt from the three′-end are extruded from RNAP as evidenced by their accessibility to RNases and crosslinking data (26, 32). RNA leave channel is formed by flap (a flexible loop of β subunit) as well as by rudder and hat loops and Zn-finger domain of β′ subunit (Fig. 1 B). Formation of a complete TH is essential for termination (33). Although it has been proposed that at some terminators Thursday formation is accompanied by forwards translocation of RNAP (34), at other terminators, TH does not shift the enzyme (35). In the latter scenario, it is hard to excogitate how, at the termination betoken, the left arm of the hairpin can reach the 9th nucleotide from the RNA 3′-cease, which is buried securely inside RNAP (Fig. ane, A and B). Annotation, that hairpin formation brings together four chains of the RNA: the iii′ proximal RNA, the double-stranded hairpin stem, and the upstream RNA.
Here, we studied the TH folding pathway in the context of the EC to sympathize how the EC affects this folding. More importantly, we wanted to investigate the consequences of any EC-imposed constraint on the folding and operation of the nascent RNA. To monitor Thursday folding, we stopped RNAP translocation at diverse distances downstream from the hairpin and measured the extent of base pairing by probing the RNA with single-strand specific RNase T1. Past utilizing a series of sequences, nosotros created a comprehensive movie of Thursday hairpin folding pathway. Our results provide an explanation of previous observations that the efficiency of terminators strongly depends on sequence context (36–39) and take broader implications for understanding co-transcriptional folding of the RNA.
EXPERIMENTAL PROCEDURES
Transcription Templates and Transcription Reactions
The transcribed sequences of the templates are shown in the figures. The promoter region was either the 71 bp sequence of the A1 promoter of bacteriophage T7 or the 121 bp sequence of the GalP1 promoter of E. coli (for tR2/T/GalP1 template of supplemental Fig. S6). The GalP1 promoter template, in which the −x element was brought to consensus, was a gift from Dr. K. Severinov. The templates were obtained past PCR and purified using a PCR purification kit (Qiagen). RNAP carrying a hexahistidine tag at the β′ subunit was purified from the RL916 strain (obtained from Dr. R. Landick) every bit described (40).
Transcription was initiated past incubation of 2 pmol of RNAP with 2 pmol of template in 5 μl of transcription buffer (TB; xx km Tris-HCl, pH seven.9, forty m1000 KCl, v one thousandthousand MgCl2, one gm β-mercaptoethanol) for 5 min at 37 °C followed by the addition of 100 μm triribonucleotide RNA primer ApUpC and 20 μk GTP, CTP, and UTP for another 5 min. For template G3S, the added nucleotides were GTP, CTP, and ATP; for template tR2/T/long the nucleotides were GTP and ATP. The procedures that follow were washed at 25 °C. The formed EC was immobilized on 20 μl of Ni-NTA-agarose beads (Qiagen) prewashed with TB. After 5 min of incubation, the immobilized EC was done five times by resuspending the beads in 1 ml of TB and brief centrifugation. The immobilized ECs were walked to the desired position of the templates by incubation with five μg NTP subsets in twenty μl TB for 3 min followed past washing. The transcript was labeled in the Ah+i and Ah+2 positions in ECh+2, in the Ah+4 position in ECh+5, h+half dozen, h+seven, h+8, h+ix by incubation with twoscore μCi of [α-32P]ATP (New England Nuclear, 3000 Ci/mmol). For termination experiments on templates G1, G1/T, G3/T, G1L/T, tR2/T, the transcript was labeled in the A position of the loop; for termination experiments on template tR2/T/long, the transcript was labeled in the twelfth C from the transcription beginning site past incubation with 40 μCi of [α-32P]CTP (New England Nuclear, 3000 Ci/mmol). The ECs were broken with RNase T1 or chased with four NTPs (as described below) and separated by denaturing urea Folio. The gels were exposed to x-ray film or scanned on Phoshoimager and analyzed by Image Quant software.
Cleavage of the RNA with RNase T1
The immobilized ECs were incubated in 50 μl TB with 500 units/ml RNase T1 (Boeringer Mannheim). X microliter aliquots were taken at the time points indicated in the figures, combined with 3 μl of phenol and vortexed immediately for 5 south. The last samples in the kinetics were washed with 1 ml of TB, volume was brought back to 10 μl, 3 μl of phenol were added. Each sample was combined with 10 μl of gel-loading buffer (50 1000m EDTA, 10 n urea) and analyzed past denaturing Page.
We previously demonstrated that using high concentrations of ribonucleases produces misleading results regarding the country of the nascent transcript in the ECs (32). This artifact occurs because the handling used to inactivate the RNase causes the disintegration of the EC before the RNase loses its action, and the RNase cleaves the transcript released from the denatured EC. However, RNase T1 does non cause this artifact when used at a concentration of 500 units/ml (32).
For the experiment with gratis RNA, ECh+5 or ECh+7 were separated by denaturing Folio, the radioactive RNA band was cut from the gel, RNA was extracted from the gel twice past incubation with TB and treated with RNase T1 as described above.
Transcription Termination
For the termination examination, the immobilized ECs were incubated with four NTPs in x μl of TB containing 300 mk KCl (except in supplemental Fig. S4) for 3 min, and then combined with gel loading buffer and analyzed by PAGE. In the experiments of Figs. 5 D and half dozen B, and supplemental Fig. S4, the immobilized ECs were incubated with 4 NTPs in the indicated conditions in 20 μl of TB, briefly vortexed and centrifuged, 10 μl of the supernatant were removed and combined with 10 μl gel loading buffer (S fraction), the remaining 10 μl of the supernatant and pellet were too combined with 10 μl of gel-loading buffer (P fraction). The fractions were analyzed by Page.
The outcome of hairpin sequence on termination efficiency mediated by alternative RNA structures. A, transcribed portions of the templates used in B–D. B, RNA-labeled ECh-nine was chased with four NTPs and analyzed by denaturing Folio. C, ECh+2 and ECh+eight were obtained using template G1L and probed with RNase T1 as in Fig. 2 B. The h+9 transcript is nowadays in lanes 7–12 considering the of the NTP cantankerous-contamination. Open and closed scissors represent susceptibility and resistance, correspondingly, of G1 to the cleavage with RNase T1 in the two potential structures. D, ECs labeled in the RNA and immobilized on Ni-NTA-agarose as described nether "Experimental Procedures" were chased with four NTPs, one-one-half of the supernatant (S fraction) and the remaining half of the supernatant and pellet (P fraction) were separately analyzed past denaturing PAGE. Complete dissociation of an EC results in equal distribution of the terminated RNA between the two fractions.
The effect of sequence context on termination efficiency mediated by culling RNA structures. A, sequences of tR2/T and tR2/T/long templates. B, transcription was performed on templates tR2/T and tR2/T/long and supernatant (South) and pellet (P) fractions were analyzed separately every bit described nether "Experimental Procedures." C, predicted secondary structure of the transcript in the EC that reached the termination point on the tR2/T/long template (ECh+7) shown both equally a sequence and as an outline. The prediction is based on the assumption that ten iii′ proximal nt (gray background) cannot participate in base pairing with the upstream part of the RNA. The complementary interactions competing with the formation of the Th are boxed. D, scheme of RNA cleavage in ECh-36, the oval symbolizes the segment of the transcript protected by RNAP from RNases, the labeled C residue is shown in greyness. E, ECh-36 shown in D was obtained using template tR2/T/long, ane-third of the complex was chased, another third was chased afterwards treatment with RNase A and launder, and the balance was chased later treatment with RNase T1 and launder.
Transcription Termination after RNase T1 Cleavage
ECh-36 obtained using template tT2/T/long and labeled in the 12th C from transcription commencement site was incubated for v min with 5 mg/ml RNaseA or with 5,000 units/ml RNase T1, done ten times with TB, then incubated with indicated concentrations of four NTPs in 10 μl of TB containing 300 mthousand KCl and analyzed past Folio.
RESULTS
Templates Used to Study Terminator Hairpin Folding
To address hairpin folding, nosotros created a series of templates mimicking the tR2 terminator of phage lambda, a well-studied intrinsic transcription terminator (33, 34, 38, 39, 41–43), which does not support forward translocation of RNAP (35). The tR2 terminator consists of a hairpin, which has a 7 bp stem and an eight nt loop, followed past an oligoU rails (Fig. ane A). Because we chose to appraise the hairpin folding employing RNase T1, which cleaves RNA on the iii′ side of unpaired Thou residues, we substituted all just one G in the left (i.eastward. the upstream) arm of the stem and in the loop of tR2 hairpin with other residues, while correspondingly irresolute the right arm so as to preserve the base pairing. In different templates, we varied the position of this single G within the left arm. The transcribed sequences of templates G1, G3, G5, and G7 are shown in Fig. ii A. We refer to the residues in the left arm of the hairpin co-ordinate to their position relative to the starting time of the hairpin sequence. In template G1, the single G, Kane, was located at the very base of the stem (see as well schemes in Figs. ii C and vii A). In template G7, the single Thou, Yard7, was the farthest one from the base of the stem and the closest one to the loop.
Probing with RNase T1 the folding of the TH in the EC. A, sequences of the templates employed to follow RNA hairpin folding during transcription. The "signal" G residues in the left arm are shown in bold with their positions indicated by subscript numerals, and the mismatched residues in G1/mis are boxed and in gray blazon. Arrows below the sequences mark the left (upstream) and the right (downstream) arms of the hairpin. B, indicated ECs were treated with 500 units/ml RNaseT1 for the time periods shown, the cleavage was stopped with phenol, the products were analyzed past denaturing Folio. 1 of the two xx-min samples was washed with TB before phenol addition (marked every bit west). The RNA was labeled in the A h+four position, except in ECh+2, which was labeled in A h+1,h+ii positions. The asterisk marks the production originating from free RNA dissociated from ECh+seven and cleaved later on Thouh+v (see also supplemental Fig. S1). Lanes 25–29, the RNA was isolated from ECh+7 as described nether "Experimental Procedures." C, RNAP was walked to the h+5 position on templates G1 and G1/mis, the RNA was extracted from the EC, treated with RNaseT1, and analyzed by Folio. The uncut RNAs have the same size but dissimilar mobility because of the issue of the potent secondary structure formed by the hairpin of the G1 transcript. The schemes represent the "perfect" and "mismatched" hairpins formed by the two RNAs, and the response of the Kone residue to the cleavage with RNase T1. Open pair of scissors symbolize susceptibility to RNase T1, closed pair of scissors symbolize resistance to the cleavage. Labeled A h+4 is also shown. D, ECs obtained using template G1/mis were treated with RNase T1 as in B.
Schematic of TH folding in the context of the EC. Meet explanations in the "Discussion." A, grayness lines symbolize the Dna, the two strands of which separate to form transcription bubble. Black/royal/crimson lines symbolize nascent RNA, which contains sequences encoding the stalk (light cherry) and the loop (imperial) of the TH. The reddish numbers one, 3, five, vii show the nucleotide positions in the left arm of the Th counting from the base of the stem. Eight iii′-last bases of the transcript base pair with template strand of the DNA in the region of transcription chimera to form RNA:Deoxyribonucleic acid hybrid. The gray oval represents RNAP and the small gray circle represents the active center of the enzyme, which coincides with the 3′-end of the RNA. The position of the upstream end of RNAP is based of footprinting data showing protection of xv nt of RNA and Deoxyribonucleic acid from nucleases, the downstream end of RNAP is placed arbitrarily and does not reverberate footprinting data. B, considering of EC interference with folding of the hairpin, small-scale RNA-RNA interactions affect termination. The dashed oval line represents a state of affairs that would arise if RNAP did not interfere with TH formation, the thick oval line represents the EC interfering with the TH formation.
All the experiments were washed with a His-tagged RNAP from East. coli immobilized on Ni-NTA-agarose beads to let stepwise "walking" of RNAP along the template (40) past alternately using incomplete sets of nucleoside triphosphates (NTPs). To stall the ECs at whatever position downstream from the hairpin, we substituted the oligoT runway of the tR2 terminator with a sequence that allowed walking in single nucleotide steps by NTP omission. In addition, this new downstream sequence provided a stronger RNA:DNA hybrid to stabilize the ECs halted downstream of the hairpin against hairpin-induced dissociation (33). We refer to the halted ECs according to the location of the RNA iii′-ends relative to the finish of the hairpin sequence. For example, ECh+5 has been halted at 5 nt downstream from the base of the stem. The templates contained the strong phage T7A1 promoter; three nt at the commencement site of transcription (AUC) were retained every bit in the wt T7A1 sequence to maintain accurate start site selection and efficient initiation.
Cleavage of the Base of the Thursday with RNase T1
First, we probed with RNaseT1 a series of ECs halted 2–9 nt downstream from the TH of template G1. In this, and in all other RNase cleavage experiments, the RNA was labeled at the three′-terminate in Ah+1, h+2 positions in ECh+2 and in Ah+4 position in ECh+v through ECh+ix. Labeling in Ah+4 position was equivalent to the iii′ end labeling for these ECs, considering 14–sixteen 3′ proximal nt are normally protected past RNAP (32). For the same reason, G residues in the right arm of the hairpin were expected non to be cleaved. Therefore, G1 residue was the only K potentially accessible by RNase T1 (see supplemental Fig. S1 for clarification). The ECs were treated with 500 units/ml RNase T1 for diverse time periods. Considering RNA hairpins return the ECs unstable (33, 44), the samples taken at the last point of each kinetics were washed to distinguish the RNA cleaved in the EC from the RNA cleaved after release from the EC.
In ECh+2 and h+5, residue G1 was extensively broken (Fig. 2 B, lanes 1–12; the supplemental Fig. S1 clarifies the results presented in Fig. ii B). This sensitivity of G1 to RNase T1 demonstrates that the final base pair of the stem was not formed in these complexes fifty-fifty though the stem-encoding sequence (and a few more nt) had been synthesized. This result shows that EC interferes with the hairpin germination. Indeed, in the free RNA isolated from ECh+v, Chiliad1 was protected by the TH from cleavage with RNase T1 (Fig. 2 C, lanes one–5). To confirm that our conditions discriminated between paired and unpaired G residues, we treated with RNase T1 transcripts isolated from ECh+5 made on templates G1 and G1/mis. G1/mis template contained substitutions that kept G1 and C2 of the left arm of the hairpin unpaired (run across Fig. ii, A and C). After 20 min of incubation of the free RNA with RNase T1, the fully complementary hairpin remained intact but the mismatched hairpin was cleaved at the One thousandi position (Fig. ii C, lanes v and 10).
In ECh+6 and ECh+7, G1 remained sensitive to RNase T1 (Fig. ii B, lanes thirteen–24), demonstrating the lack of base of operations pairing in these complexes. Tr2 hairpin destabilizes ECh+7 nearly strongly of all downstream ECs (Ref. 33 and supplemental Fig. S2, A and B). Correspondingly, in ECh+7, we observed two products of cleavage: one product was removed by washing the beads while the other remained bound to the EC (Fig. ii B, lanes 23 and 24). The former product resulted from the cleavage of the RNA released from the unstable ECh+seven. This released RNA was cleaved at Gh+v, as confirmed by the cleavage of the free h+7 RNA in lanes 25–29 (see also supplemental Fig. S1).
In ECh+8 and ECh+9, by contrast, Kone became resistant to cleavage (lanes xxx–41). In these complexes, G1 can be protected by formed hairpin stem, by RNAP, or by both. To distinguish amid these possibilities, we tested cleavage of G1 in the ECs obtained on G1/mis template. In ECh+8 obtained using this template, Gone was resistant to cleavage (Fig. 2 D, lanes 11–15), presumably because the bottom part of the hairpin was brought inside RNAP despite the fact that it could not form base pairs. We speculate that in ECh+8 obtained using template G1, which generated fully complementary TH, the hairpin was formed fully inside RNAP. In other words, the protection of the base of the Th in ECh+8 (Fig. 2 B, lanes 30–35) was caused by both hairpin stem formation and by RNAP. We call back that the bottom of the stem was brought within RNAP during a major structural transformation, which occurred in the EC when the enzyme translocated from the h+7 to the h+8 position. Nosotros make this conclusion because, dissimilar in ECh+8, in ECh+vii formed on the mismatched template, M1 was sensitive to RNaseT1 (Fig. 2 D, lanes 6–10). Nosotros tested the formation of the acme of the hairpin in the side by side experiment.
Probing the Entire Left Arm of the Hairpin
To probe base of operations-pairing at the other positions of the stem, nosotros tested the RNase sensitivity of a series of complexes obtained using templates G3, G5, and G7 (Fig. 3). In ECh+ii and ECh+five, residues 10003, K5, and Gvii were cleaved, indicating that non a unmarried base pair of the hairpin was formed equally RNAP transcribed upwards to five nt downstream from the stem. In ECh+6, an unexpected pattern of cleavage was observed: Grand5 and Gvii were cleaved just G3 was non. This upshot suggests an unusual RNA construction in ECh+6, which is addressed in the next department. In ECh+7, Yardiii, One thousandv, and G7 were protected from cleavage suggesting the germination of the upper portion of the hairpin. Note that the RNA released from ECh+vii was cleaved at position Gh+v, every bit expected. In ECh+eight, none of the G residues in the left arm were broken, indicating full hairpin formation. All the same, even in ECh+8, the EC yet impedes the hairpin folding every bit will be discussed later on.
Probing of Thousandiii, G5, and G7 hairpin positions with RNase T1. The ECs were obtained using templates G3 (A), G5 (B), and G7 (C) and probed with RNaseT1 equally described for Fig. ii B. Asterisks marker the cleavage products originating from gratuitous RNA dissociated from the unstable ECh+6 and ECh+7.
Importantly, the protection of the left arm in ECh+eight was not acquired by backtracking of RNAP to that region (45) considering in ECh+8 obtained on tR2 template, which encoded Yard residues in the loop of the hairpin, the G residues of the loop were sensitive to RNaseT1 (supplemental Fig. S2, A and C).
Hairpin Formation in ECh+6
In ECh+half dozen, G3 was protected from RNase T1 (Fig. 3 A) while M1, G5, and Gvii were cleaved (Figs. ii B and iii, B and C), which suggested an unlikely RNA conformation in the EC depicted in Fig. 4 A. Still, the formation of a structure alternative to the Thursday by the portion of the RNA extruded from the EC could also explain this upshot. In search of such a construction, we computationally mimicked co-transcriptional folding using four series of RNA sequences corresponding to each of the iv templates (46). Each RNA began at the get-go site of transcription, but the three′-ends ranged from position h-7 to h0. While no strong alternative structures were establish for three of the templates, G3 produced ii structures with similar complimentary energies, when four downstream nt of the hairpin were excluded from the folding (i.eastward. when the 3′-stop of the folded sequence was at h-4, Fig. 4 B). The first structure was a predecessor of the Th; in the 2nd, more stable structure, G3 was paired with a C residue that would be located in the loop of the TH. This situation would ascend if the EC prevented the x 3′-proximal nt of the transcript from participation in the folding. To see if the predicted culling structure explains the lack of RNase T1 cleavage in ECh+6 obtained using template G3, we changed the sequence of this template in the means that selectively weakened the alternative structure (templates G3S and G3L (named then for substitutions in the stalk and the loop); Fig. 4, C and D and supplemental Fig. S3). In h+6 complexes obtained using these templates, 1000iii regained sensitivity to RNase T1, indicating that information technology was not paired with the correct arm of Th. ECh+7 obtained on either template efficiently dissociated and the free RNA was cleaved at Gh+five balance. In ECh+8, there was no cleavage, which confirmed the formation of the hairpin in this circuitous. We concluded that the predicted alternative structure accounts for the RNase resistance of residue Thousandiii in ECh+6 obtained on template G3 in the experiment of Fig. iii A.
Hairpin germination in ECh+6. A, scheme represents the RNA structure in ECh+six based on RNase T1 cleavage of this complex formed on templates G1, G3, G5, G7. The grey oval symbolizes RNAP. B, RNA secondary structures predicted for the ECh+half dozen transcript formed on template G3 based on the supposition that ten iii′ proximal nt (shaded by gray background) either practise not participate or participate with difficulty in base-pairing with the upstream function of the RNA. Open and closed scissors represent susceptibility and resistance, correspondingly, of Giii to the cleavage with RNase T1 in the two structures. C, sequence of the transcript in ECh+vi made using template G3S and its predicted structure. D, ECs were obtained using template G3S and probed with RNaseT1 equally described for Fig. two B. The bands in lanes 7–12 are, from top to bottom, (i) h+7 uncut, (2) h+half-dozen uncut, (3) the product of the cleavage of free RNA dissociated from ECh+7 cut at One thousandh+5 (marked by the asterisk), (four) the product of the cleavage of the nascent transcript in ECh+vi cut at K3. The h+half-dozen transcript is present in these lanes considering of its inefficient elongation.
Alternative Structures Affect Termination
Taken together, the results of RNase T1 cleavage of ECs halted downstream from the hairpins modeled later on the tR2 terminator suggest that the Thursday formation starts when RNAP transcribes at vii nt downstream from the stem and completes at 8 nt downstream from the stem (as shown schematically in Fig. seven A). These positions represent two termination points of the tR2 terminator. The Th formed during transcription of template G1 supported efficient termination when the template was modified to include an oligoT track (template G1/T, Fig. 5, A and B, lanes 1 and ii). Fifty-fifty in the absence of an oligoT rail (template G1), RNAP terminated transcription at positions h+7 and h+eight and released RNA, although with a much reduced efficiency (supplemental Fig. S4). The power of RNAP to terminate accurately in template G1 validates our using this template to address TH germination in the experiments described above.
The results obtained with ECh+vi betoken to the ease, with which the exposed portion of RNA forms secondary structures that compete with the Th. This tendency revealed in static halted ECs should strongly affect termination efficiency in a dynamic setting. We detected competing structures in halted ECs formed using template G3 but non G1. Correspondingly, during uninterrupted transcription in the presence of all iv NTPs, termination was less efficient on G3/T template than it was on G1/T template (Fig. 5, A and B). Still, the G3 hairpin was slightly weaker than the G1 hairpin, a departure that could contribute to reduced termination.
To further test the hypothesis that a transient culling RNA construction can affect termination efficiency, we designed another hairpin, G1L, that had the same calculated free free energy of formation every bit the G1 hairpin just contained two substitutions in the loop (template G1L, Fig. 5, A and C). In ECh+8 obtained using the G1L template, an alternative secondary structure, in which the Thousand1 residue is non base of operations-paired, is possible when ten nt of the 3′-end are excluded from folding (Fig. 5 C). Indeed, we found that residue G1 was RNase-sensitive in ECh+8 obtained on G1L, unlike its resistance in template G1 (Fig. two B, lanes 30–35, and Fig. 5 C, lanes vii–12). This issue shows that despite the fact that the Th can grade in ECh+8, the EC still impedes the folding. In ECh+nine, the RNA was resistant to RNase T1 on both templates because a greater portion of the hairpin is gratuitous from RNAP, and this is expected to aversion the alternative construction. In agreement with the beingness of the alternative construction competing with the Th in the static atmospheric condition, termination, probed in dynamic atmospheric condition, was much weaker on the G1L/T template than on G1/T template (Fig. five, A and D).
Sequence Context Affects Termination Efficiency via Weak Transient Competing Interactions
The propensity of the left arm of the hairpin to participate in competitive interactions makes termination highly dependent on sequence context, even when the Th itself is not changed at all. In template tR2/T, the tR2 terminator is closer to the get-go site of transcription than in template tR2/T/long: the hairpin sequence begins at 3 and 34 nt downstream from the offset site, respectively (Fig. six A). In 100 μthousand NTPs, 17% of the ECs read through tR2 terminator on the short template, as compared with 52% on the long template; in 10 μ1000 NTPs, the lower concentration that increases termination, the read through is 2 and 13% (Fig. half-dozen B). The add-on of an oligonucleotide complementary to the start xxx nt of the transcript increased termination efficiency on the tR2/T/long template (supplemental Fig. S5), which pointed to the existence of a secondary construction competing with the Th.
Computer-assisted folding of the terminated RNA (ending at h+7 position) did non reveal any construction strong enough to compete with the Thursday (46). However, when x 3′ proximal nt of the transcript were excluded from the folding, the v′-cease of the RNA showed a potential to base pair with the nearly upstream residues of the terminator stem (Fig. 6 C). To examination if these interactions indeed inhibited termination on the tR2/T/long template, we walked RNAP to 20 nt from the start site to form ECh-36. We treated the complex with either pyrimidine-specific RNase A, which cleaved off three 5′ last nt, or with G-specific RNase T1, which cleaved off six 5′ terminal nt (Fig. half dozen, D and E). The rest of the transcript was protected from the RNases by RNAP (32). After washing off the RNases, nosotros chased the truncated circuitous. The removal of merely iii 5′ terminal nt significantly increased the relative corporeality of terminated product, and the removal of six nt caused a farther increment (Fig. 6 E).
Give-and-take
Fig. 7 A depicts the pathway of Thursday formation in the context of the EC based on RNase T1 cleavage patterns. In ECh+five, RNase T1 cleaved the RNA at all G residues of the left arm of the hairpin. In ECh+6, residues G1, K5, and Gvii were besides cleaved. Residue Chiliadiii was cleaved in two of the three templates (G3L and G3S), simply not in template G3, where it was protected by an alternative secondary structure. Thus, in ECh+5 and ECh+6, the TH had non yet formed.
In ECh+vii, treatment with RNase T1 revealed two fractions, which reflected the pronounced trend of the TH to dissociate the complex. In 1 fraction of ECh+7, Gi but not 1000three, One thousand5, and Gvii were cleaved by RNase T1, suggesting the formation of the "top" part of the hairpin. The completion of the hairpin is prevented by both RNAP protein and RNA:DNA hybrid. The other fraction of ECh+seven was not cut at whatever of these Gs but it dissociated in the course of the handling with RNase T1. We remember that in this fraction, full Th formed and it instantly dissociated the EC. Because of this exceptional instability of ECh+vii (supplemental Fig. S2B), the full hairpin could not be detected with RNase T1 in this complex.
ECh+eight was the outset complex where protection of all the G residues was detected at most templates, revealing the formation of the full hairpin. At some sequences, RNAP however impeded hairpin folding and enabled formation of alternative structures.
The proposed pathway of Th formation clarifies important details of the intrinsic termination mechanism. First, our information show that in ECh+seven (the position corresponding to the start termination point) the hairpin is partially formed in a way that mimics a pausing hairpin (Fig. 7 A). This finding supports the idea that hairpin-dependent pausing is a part of the termination process (33, 47).
Second, the results of G1 cleavage in ECh+seven and ECh+8 on G1/mis template suggest a major structural rearrangement of the EC occurring betwixt these two positions. Current models of termination imply that hairpin formation destabilizes the EC by causing forward translocation of the RNAP, shearing of the RNA:DNA hybrid, or major changes in RNAP conformation (33, 34, 48, 49). In the ECs formed on G1/mis template, the sequence allowed formation of five base pairs at the superlative of the stem only did not permit germination of the base pairs at the bottom of the stem (Fig. 2 D). As expected, G1 was cleaved past RNase T1 in ECh+seven, only, surprisingly, was resistant to the cleavage in ECh+8. This effect suggests that in ECh+8, the lesser of the stem is brought inside RNAP past the force of partial formation of the TH. X-ray structures of RNAP prove that the width of the RNA leave channel remains the aforementioned forth the whole channel (28). In this case, the protection of the mismatched base of the stem in ECh+8 signifies that the TH formation causes major EC rearrangement, which may be necessary for RNA release. These rearrangements can involve shifting of RNAP domains that collide with the hairpin (come across Fig. ane B), or opening of the entire RNAP clamp. Before, we found that a complete hairpin that formed in ECh+8 caused melting of the next base-pairs of the RNA:Dna hybrid (33). Shortening of the hybrid destabilized the EC and besides contributed to termination of transcription. We propose that similar rearrangements occur in ECh+7 as well, but they cannot be detected because of immediate dissociation of the complex upon completion of the hairpin.
Our finding that the EC interferes with the germination of the Th tin can explain some previous observations. All termination models agree that hairpin germination is necessary and sufficient for cessation of RNA synthesis and RNA release, if the hairpin is followed by a U-rich sequence. The stability of the hairpin only not its particular sequence is believed to touch on termination efficiency. This view is supported by the fact that a smashing multifariousness of the hairpins office in E. coli as the components of intrinsic terminators (50). At the same time, it was reported that some mutations that did not modify or fifty-fifty increased the stability of the hairpin decreased termination efficiency (38, 39). These results suggested that the sequence of the hairpin is important for elongation-termination choice, due to specific interactions of the hairpin with RNAP (38). In addition, it was constitute that the same terminators controlled by dissimilar promoters function with different efficiency (36, 37). These results led to models, in which promoter-proximal sequences affected termination capacity of RNAP past modifying the conformation of the enzyme (36, 37). However, such RNAP conformations accept never been specifically characterized. The authors (36, 37) considered an alternative explanation, in which some segments of promoter-proximal sequences linked to these various promoters anneal to the left arm of the Th because of extensive homology to it past a machinery like to transcription attenuation in bacteria (xi). However, computer-assisted folding did not discover the expected base pairing in vast majority of the constructs. EC interference with folding of the hairpin, reported hither, significantly broadens the range of base pairing opportunities that can touch on termination (Fig. 7 B). On many sequences, in the absence of EC interference, no construction strong enough to complete with the TH can course (Fig. 7 B, top scheme). Notwithstanding, since RNAP sequesters the correct arm of the TH, the left arm of the hairpin is highly susceptible to modest competitive interactions, which delay the germination of the TH (Fig. 7 B, bottom scheme). Such interactions can involve RNA sequences located significantly upstream of the Thursday simply brought close to it by overall folding of the transcript. As a result, the Thursday is not formed when RNAP transcribes the point of termination, and termination is suppressed. Such subtle RNA-RNA interactions could explain in many cases the dependence of termination efficiency on hairpin sequence and on the sequence context. In understanding with this model, unmarried molecule assays showed that application of a weak force pulling the RNA 5′-cease away from RNAP increased termination efficiency (35). This forcefulness was not sufficient to pull the EC apart or to unfold the TH, and it was proposed that information technology unfolded smaller secondary structures that compete with the TH. We excluded the possibility that initiation mode affects RNAP power to end by showing that RNAP initiating at two different classes of promoters has the same termination backdrop (supplemental Fig. S6).
Our results permit making predictions of terminators' efficiency depending on their structure. These predictions are based on our conclusion that the timing of TH folding and transcription of the termination point should be strongly coordinated then that consummate hairpin is formed by the fourth dimension RNAP transcribes the termination point. Increasing the size of the loop should decrease termination efficiency, because it would enable the subtle interactions with the upstream RNA competing with the Th. For the same reason, terminators with longer stems tin be less efficient. On the other mitt, the protein interference with hairpin folding in RNA exit aqueduct defines the minimal size of TH stem (half dozen–7 bp) because the kickoff base pair tin can form only when both complementary RNA bases exit the aqueduct. Therefore, longer stems could be more efficient since the hairpin can nucleate well before termination point is transcribed to ensure the timely completion of the TH. All the same, it remains unclear how a hairpin tin can cause termination at broadly distributed multiple sites downstream from the stalk equally was reported in at least one case (51).
X-ray crystallography demonstrated that pro- and eukaryotic RNAPs have remarkably similar structural features (52) implying common mechanisms of their performance and regulation. RNAPII terminates in vitro at an intrinsic bacterial terminator (33) suggesting that many rules defining stability of the EC and folding of nascent RNA are common for pro- and eukaryotic RNAPs. We propose that EC interference with transcript folding can affect the formation of functional RNA in both pro- and eukaryotes and tin can influence such regulatory processes as co-transcriptional binding of proteins to RNA, transcription pausing, termination, and RNA splicing.
Supplementary Cloth
Acknowledgments
We thank Mikhail Kashlev and Robert Weisberg for providing facilities for inquiry in their laboratories, Robert Weisberg for reading the manuscript and critical communication, Arkady Mustaev for comments and help with Fig. 1B, Michael Lichten for suggestions on the manuscript, Robert Landick for strain RL916, Konstantin Severinov for the GalP1 template, and Shelley Lloyd for participating in the early stages of this project.
*This piece of work was supported, in whole or in part, past the Intramural Research Programme of the National Cancer Institute, Center for Cancer Research, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.
The on-line version of this article (bachelor at http://world wide web.jbc.org) contains supplemental Figs. S1–S6.
iiiThe abbreviations used are:
- RNAP
- RNA polymerase
- EC
- elongation complex
- Th
- termination hairpin
- TB
- transcription buffer
- nt
- nucleotides.
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