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Which factors besides the thermodynamic stability are important for the hairpin in intrinsic transcription termination?

Which factors besides the thermodynamic stability are important for the hairpin in intrinsic transcription termination?



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Intrinsic termination (rho-independent) relies on a stable hairpin with a subsequent uridine repeat. The common explanation on how these sequences cause the termination of the transcription are based on the thermodynamic stability of the sequence. The GC-rich stable hairpin together with the destabilizing U-repeat supposedly destabilize the binding of the polymerase enough to cause it to disassociate from the template.

What I'm looking for are specific requirements on the termination sequences that are not based on thermodynamics.

  • Are there any specific requirements on the hairpin sequence beyond a certain thermodynamic stability.
  • Are there any loop variants that are known to reduce the termination efficiency?
  • Does the shape of the hairpin, e.g. any kinks or bulges in it, have an influence on termination efficiency?

The typical requirements I read are 5-14bp and GC-rich, and I'd like to know if there are any more specific requirements, especially ones related to the structure and not the stability of the hairpin.


The RNA hairpin transcription terminator in prokaryotes is terminated by the GC rich hairpin followed by 4+ uracil bases as you describe it.

The original reference describing the hairpin, described the sequence requirements as being for the thermodynamic stability of the hairpin, but the protein nusA has also been found to stimulate termination.

You might think that there would be some protein that would precisely bind this motif and mediate the termination, but at least as far as I can find, it appears that the hairpin clogs up the polymerase itself, causing termination. This review linked here has some nice pictures of the RNAP bindig pocket model.


Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

RNA polymerase (RNAP), like many cellular processors of information in DNA and RNA, is a complex macromolecular machine whose multiple structural modules and domains undergo poorly understood conformational changes that mediate information processing. We investigated the role of one such mobile module, the polymorphous trigger loop (TL) of RNAP, in intrinsic transcription termination by bacterial RNAP. The TL folds into a helical hairpin to promote RNA synthesis, but also is proposed to aid termination. By separating effects of the TL and of TL variants on termination from effects on RNA synthesis, we established that TL flexibility, not the helical hairpin conformation, facilitates rearrangements of RNAP leading to termination. Our results illustrate how kinetic assays can help dissect complex macromolecular machines.


Abstract

The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli . We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies ( r = 0.9, n = 31) better than models trained on all terminators ( r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.


DISCUSSION

Currently, our understanding of the kinetics of transcript elongation trails our understanding of the mechanics. Although cryo-EM structures of paused TECs gave insight into the mechanisms of distinct pausing signals, these static snapshots cannot reveal the kinetics of pause states. For kinetics, we rely on single molecule experiments that reveal the dynamics of transcript elongation in conjunction with modeling that can decompose the overall dynamics into contributions from individual components. A Brownian ratchet model accurately reflects the rate and the force-dependence of the processive elongation observed in experiments. However, the kinetics of paused states are less clear: (i) in different proposed models, the ePEC are represented as either on-pathway or off-pathway states, and the models do not seem to completely fit the mechanistic origin of the ePEC (ii) kinetics of the bPEC are well characterized, but fit-determined parameters vary according to experimental constructs (iii) and finally, experiments to analyze the kinetics of the hsPEC are difficult. Overall, the difficulty in characterizing the kinetics of the paused states arises from distinguishing the observed pauses experimentally, especially for the elemental and hairpin-stabilized PECs which cannot be distinguished translocationally in single-molecule transcription assays. Even for bPECs, which are characterized by reverse motion of TECs, efficient identification requires base-pair resolution and high signal-to-noise ratio experiments that are difficult to achieve in most laboratories.

The Brownian-ratchet model predicts the configuration of a transcription bubble to be a subtle but critical element that could significantly affect the kinetics. Nucleotides 1–2 positions proximal to the catalytic site and the unpaired nucleotides at the edges of the bubble are important, because they chiefly determine the relative stability of the pre- and post-translocated states, and hence the possibility of entering a paused state. Many models employ a fixed-length transcription bubble and a fixed-length DNA/RNA hybrid ( 40, 77, 79). These assumptions conflict with changes in the size of the transcription bubble detected experimentally and may introduce thermally unfavorable bubble configurations that might change spontaneously. A statistical mechanics approach was implemented in some model constructs to account for the variation in bubble/hybrid size ( 72).

The Brownian-ratchet model of RNAP transcription was proposed 30 years ago and has been subsequently refined. However, a significant defect of the model is a probable over-simplification of the real transcription mechanism, such as neglect of a potential allosteric nucleotide binding site that the elongation complex may contain, as proposed by Foster et al. ( 80). In addition, the effects of transcriptional modulators are overlooked in most models, and none address heterogeneity of pausing among species. For example, in E. coli, NusG is an anti-pausing factor that could stimulate forward translocation and prevent RNAP backtracking, while in Bacillus subtilis, NusG induces pausing by shifting RNAP to the post-translocation register ( 26, 81–84). Another example is that E. coli RNAP recognizes a well-characterized hairpin-stabilized his pause site, while B. subtilis RNAP and mammalian Pol II do not respond to this hairpin-mediated signal ( 26, 85).

Despite the limitations, models of transcript elongation have produced new insights about the kinetics of mechanistically identified paused states. For example, in modelling the distribution of pause times of bacterial TEC, Janissen et al. identified three interconnected pause states, two of which appear to be backtracked PECs. They found that the recovery from one occurs 20 times slower than that from the other and cannot be accelerated by cleavage factor GreB ( 57). Therefore, they postulated that a bPEC could undergo conformational changes to enter a longer-lived RNA-cleavage-resistant state. Douglas et al., by comparing models with and without the intermediate state, backtracking, hypertranslocation and RNA folding, found that these factors are not necessary for predicting the locations and frequencies of pauses ( 77). Thus, they concluded that occurrence of pauses is chiefly facilitated by the relative stability between the pre- and post-translocated states, while the off-pathway events only serve to extend the pauses.

In the future, we hope to see a unified kinetic model of transcript elongation that concurs with our biochemical understanding of the effects of other factors on paused complexes and predicts the experimentally characterized pause sites and pause frequencies. An immediate difficulty is the construction of distinctive models for the hsPEC and other mechanistically similar states and experimental methods with which to detect them. Moreover, such models must be consistent with the effects of transcriptional modulators that accentuate particular mechanisms of elongation and pausing from prokaryotes to eukaryotes.


Rho Factor

A Role for NusG in Termination

Although Rho alone can cause termination of transcription in a purified system under certain artificial conditions, another protein, NusG, is needed for Rho to function in its normal cellular context. NusG is a 19-kDa protein that can bind to both RNA polymerase and Rho. It is also known to accelerate the rate of transcriptional elongation and the rate with which Rho releases transcripts from arrested complexes. It becomes essential in vitro under conditions where the action of Rho is kinetically limited Rho does not function very well with some transcripts if the time interval between interaction with the RNA and passage of RNA polymerase through the tsp region is very short. Because NusG can bind to both Rho and RNA polymerase, it could serve as a bridge to facilitate the location of rut sites by Rho on nascent transcripts. However, this role has been brought into question by some recent evidence that Rho can bind to RNA polymerase well before a nascent transcript emerges. An alternative (or possibly additional) role for NusG could be to alter the response of RNA polymerase signals that cause it to pause during elongation and to release a transcript at a pause site in response to Rho factor.


Ancient RNA stems that terminate transcription

Multi-subunit RNA polymerases are the enzymes that perform transcription in all living organisms and that have emerged before the divergence of domains of life. The structures of catalytic cores and their functions during elongation step of transcription cycle are very similar for all multi-subunit RNA polymerases. In contrast, the mechanisms for terminating the RNA synthesis have seemingly diverged in modern RNA polymerases. However, the recent finding that, much like during bacterial transcription, RNA secondary structure is involved in termination by eukaryotic RNA polymerase III (pol III), suggests that RNA-dependent termination may have emerged before the divergence of bacterial and archaeal/eukaryotic RNA polymerases. In the case of pol III, the terminating RNA secondary structures are not dedicated hairpins, but are formed by the bodies of highly structured transcripts, which are clearly the remnants from the RNA–protein world. Here I discuss the similarities and differences of RNA-dependent mechanisms of termination of transcription by bacterial RNA polymerase and pol III.

Termination is an obligatory event that causes extremely stable transcription elongation complex to disassemble at the end of the gene with the release of RNA polymerase (RNAP) and the transcript from the template DNA. Termination is required for proper expression of neighboring genes, maturation and export of transcripts (in eukaryotes), recycling of RNAP, and clearing the template for subsequent transcription. Though mechanisms involved in transcription elongation are very similar for bacterial RNAP, archaeal RNAP and pol I, pol II, and pol III from eukaryotes (plant-specific RNAPs IV and V are not discussed here), the mechanisms of transcription termination seem to be strikingly different (see below). However, recently, we showed that, similarly to termination in bacteria, termination by eukaryotic pol III involves formation of RNA secondary structure, 1 suggesting that the RNA-dependent termination may have been the primordial mechanism used by the common ancestor of multi-subunit RNAPs.

In bacteria, destruction of the elongation complex during termination is facilitated by a dedicated > 7 base pairs-long G:C-rich RNA hairpin that folds behind RNAP. 2 , 3 Termination by pol III is also caused by RNA secondary structure. 1 However, in this case, the RNA secondary structure comes from the body of the synthesized RNA. Pol III transcribes genes of structural and catalytic RNAs (5S, SRP, RNase MRP, RNase P, U6 RNAs, tRNAs), which, as per their functions, have extensive secondary/ternary structures. The terminating RNA stem on these genes can be formed by distant parts of the transcript, such as the acceptor stem of the tRNA, formed by the very 5′ and 3′ proximal parts of the molecule ( Fig.ꀚ ).

Figureਁ. RNA secondary structure-dependent termination of transcription. (A) Scheme of the tRNA secondary structure. Note that both acceptor stem and T㲌 stem-loop can serve as termination secondary structures depending on the extent of pol III backtracking on the oligoT signal (see text). (B) Nucleic acids scaffold in the elongation complex (pdbid: 2PPB). 14 Mg 2+ ions of the active center are shown as red spheres. Template and non-template DNA are black and dark blue, respectively RNA is red. A pol III transcript, represented here by tRNA molecule (pdbid: 1EHZ), 15 folded at the distance sufficient for termination, is color coded as in panel A (with loops in pink). Note the interference of 5′ end nucleotides of the tRNA with the template DNA bases at positions 8th and 9th in the RNA𠄽NA hybrid. The real orientation of the folded tRNA relative to the hybrid may be different. More base pairs of the hybrid could be melted by the folded tRNA due to the collision of tRNA with protein domains, which could exert a pulling force on the hybrid. (C) Interference of the folded tRNA (pink) termination structure with domains of RNAP (cyan ribbon). β flap, β’ zipper, and β’ lid are shown as khaki, magenta and blue spheres, respectively. Relative orientation of tRNA as in panel B. The real orientation may differ, leading to collisions with even more domains of RNAP.

Despite this apparent difference, the mechanisms of termination between bacterial RNAP and pol III appear to be very similar. As seen from Figureꀛ , the 5′ end (5′ end shoulder of the acceptor stem) of the folded tRNA interferes with the template DNA strand in the RNA𠄽NA hybrid. This may shorten the hybrid, which is the major determinant of the stability of the elongation complex, to a critical length of 7 bp (from the RNAP active center), when the elongation complex loses its stability. 4 If the RNA stem is just 1 bp shorter, permitting 8 bp hybrid, the termination becomes much less efficient, 1 consistent with observations that elongation complexes with such hybrid length are stable. 4 A very similar situation was observed for termination by bacterial RNAP. 5 , 6 Note that shortening of the RNA𠄽NA hybrid may also happen as a result of a sterical clash of the RNA stem and the rear end of the elongation complex, which may lead to a force pulling on the RNA in the hybrid (rather than interference of the stem with the RNA𠄽NA hybrid), as was shown for bacterial termination. 5 However, the collision of the forming acceptor stem (in the case of tRNA) with structural elements of the elongation complex may play a more critical role in destabilization of the complex, as was proposed for bacterial termination. 6 , 7 A termination stem/hairpin would sterically clash with at least β flap, β’ zipper, and β’ lid ( Fig.ꀜ ). 7 Furthermore, the interference with the 5′ end of the RNA𠄽NA hybrid (via displacement or pulling) would lead to disruption of multiple protein–hybrid contacts, such as the ones with β’ rudder, which stabilize the elongation complex. 7 , 8

The ubiquitous hairpins that form during elongation cannot destruct the actively transcribing elongation complex. 2 For termination to occur, RNAP has to pause, allowing the termination hairpin to form. 6 Besides the similarities and clear involvement of the RNA secondary structure in termination, there are some noticeable differences in pre-termination pausing between bacterial and pol III enzymes. The most apparent difference is the role of oligoT (in non-template DNA strand) tract downstream of RNA secondary structures. In bacteria, the oligoT tract serves to briefly pause RNAP by causing short backtracking, which allows sufficient time for the hairpin formation. 6 , 7 In addition, the U:A RNA𠄽NA hybrid in this paused complex is required for efficient complex destruction, apparently due to its relative instability. 2 , 5 During pol III termination, oligoT tract also pauses transcription and forces pol III into backtracking. However, in this case, backtracking appears to be almost irreversible and, if continues beyond four to five nucleotides, leads to catalytic inactivation of pol III. 1 In a backtracked complex, addition of NMPs is blocked since the 3′ end of RNA has disengaged from the catalytic site. However, quite surprisingly, the highly efficient RNA hydrolysis activity, which in pol III is stimulated by the C11 subunit and would rescue the backtracked pol III by restoring the 3′ end in the active center, is also inhibited. 1 Switching off of the cleavage activity could be explained by sterical replacement of C11 from the active center by the extruding 3′ end of backtracked RNA. Such a complete catalytic inactivation, in contrast to transient pausing in bacteria, commits the elongation complex to termination.

Another unusual property of termination of pol III transcription is the insensitivity to the RNA𠄽NA hybrid sequence. Backtracking on the oligoT signal can shift pol III backward as far as

12 bp, replacing the U:A-rich hybrid in the elongation complex with a sequence that precedes it in the gene. 1 This, however, does not affect destruction of the complex by the hairpin, suggesting a generally lower stability of the pol III elongation complexes as compared with complexes of bacterial RNAP or a lesser importance of hybrid shortening during pol III termination. Extensive backtracking highlights yet another unique feature of termination by pol III. In contrast to bacteria, where the terminating RNA hairpin has to form at a strictly defined distance from the active site of RNAP, 9 the RNA stem of pol III transcripts can be as far as 12 nucleotides upstream of the oligoU stretch. 1 Accordingly, in genes transcribed by pol III, this distance varies from 0� nucleotides (in S. cerevisiae). 1 On some genes with short distances, pol III may use for termination not the nearest but the second nearest RNA secondary structure of its highly structured transcripts, such as T㲌 stem-loop that precedes the acceptor stem in tRNA ( Fig.ꀚ Fig. S8 in ref. 1 ). Such flexibility possibly increases the chances for efficient termination by pol III if the ultimate RNA hairpin fails to fold for some reason. In the case of bacterial RNAP, extensive backtracking on a termination signal would not be beneficial given that the majority of transcribed genes are coding for mRNAs, which are unlikely to have secondary structures just behind the dedicated termination hairpins.

Based on the structural similarities between the mechanisms of termination by bacterial RNAP and eukaryotic pol III, the requirement for the RNA stem for destruction of the elongation complex and the distance between this stem and the RNAP active center, it can be hypothesized that the RNA stem-dependent termination of transcription emerged before divergence of bacteria and archaea/eukaryotes. Pol II was shown to be able to terminate transcription in RNA hairpin-dependent manner in vitro, 5 and archaeal RNAP may require RNA stem folding for termination. 1 , 10 Pol I is also known to efficiently terminate synthesis of its highly structured transcript in a factor-independent manner in vitro. 11 Together, these observations are consistent with the possibility that RNA hairpin-dependent termination may have been the primordial mechanism used to stop transcription in the Last Universal Common Ancestor (LUCA), although later it may have been replaced by the protein factors. The early emergence of RNAP puts it at the stage of evolution that was dominated by ribozymes and structural RNAs (such as ribosomal RNAs or tRNAs that survived till the present days) characterized by extensive secondary structures. It seems reasonable to suggest that transcription termination caused by the structure of the transcript would be beneficiary for the LUCA destruction of the elongation complex caused by a fully folded functional RNA would provide a simple factor-independent mechanism for termination of transcription right at the end of the gene. Furthermore, RNA secondary structures affect only paused/stalled elongation complexes (irrespective of the sequence of the duplex), possibly also providing a mechanism for displacement of prematurely stalled complexes.

Though the RNA stem-dependent termination may have been the ancestral mechanism, it apparently has been supplemented and/or displaced by protein-dependent mechanisms after the divergence of multi-subunit RNAPs. Bacteria, in addition to the hairpin-dependent intrinsic termination, acquired protein-mediated termination that uses an RNA helicase ρ or a DNA translocase Mfd to destruct the elongation complex. Pol I and Pol II have acquired mechanisms involving RNA exonucleases and/or an RNA helicase, 12 , 13 which seemingly replaced RNA-dependent termination. It is likely that the archaeal RNAP may also require accessory termination proteins, and pol III may have a protein-assisted back-up mechanism in addition to the RNA-dependent termination. The two possible reasons for the emergence of new termination strategies are the growing demands for the efficiency of transcription and, as a result, for its termination and a shift from highly structured RNAs to protein-coding mRNAs that are largely devoid of regular secondary/ternary structures that could facilitate termination.


CONCLUSIONS

The E.coli genome consists of more than 2200 annotated TU ( 18, 39). Using the RNAMotif algorithm ( 21), in combination with descriptors including structure and sequence constraints and a thermodynamic scoring system, putative rho‐independent terminators have been identified for 1075 of the annotated genes and operons. Putative terminators for all annotated rRNA operons and small non-coding RNAs as well as for more than 64% of tRNA TU were found. This suggests that the transcription termination of RNA genes occurs mainly via rho‐independent mechanism or via combination of both rho-independent and rho-dependent mechanisms as was reported for rrnG operon ( 40). Putative terminators were found for half of the protein encoding ORFs suggesting that both rho-dependent and rho-independent mechanisms participate equally in transcription termination of protein-encoding TU as was predicted ( 1). We have also predicted the existence of an additional 700 putative termination signals in NCR further than 60 nt downstream from the annotated genes. These numbers are in agreement with the excess number of promoters predicted in the E.coli genome ( 39). Sequences between predicted orphan promoters and terminators in NCR may be candidates for as yet undiscovered genes.


Translation chart can result in this reason that termination in transcription termination mechanisms for instance, the random priming method of rna

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Flow cytometry data were generated within the Flow Cytometry and Cell Sorting Facility in Ashworth, King’s Buildings at the University of Edinburgh. The facility was supported by funding from Wellcome and the University of Edinburgh.

  1. ^www.addgene.org
  2. ^http://parts.igem.org
  3. ^www.addgene.org/kits/mccormick-cyanogate
  4. ^www.addgene.org/51950
  5. ^www.addgene.org/58495
  6. ^http://unafold.rna.albany.edu/?q=mfold

A DNA-binding site for RNA polymerase at which gene transcription can be initiated.

A reaction that occurs at terminator sites on the DNA template and that stops transcription by dissociating the transcription complex and releasing the nascent RNA into solution.

A stretch of 12–14 bp that is melted within the dsDNA as a consequence of transcription-complex formation. The transcription bubble exposes the DNA template strand that is then copied by RNA polymerase to form complementary RNA.

The double-stranded, 8–9-bp helix that forms within the transcription bubble when the 3′ end of the nascent RNA transcript interacts with the complementary sequence of the template.

(σ factor). A promoter-specific transcription factor in prokaryotes that binds to core RNA polymerase and directs the binding of the resulting holoenzyme to a specific promoter or family of promoters.

A measure of the accuracy with which the template sequence is copied into the complementary nascent RNA or DNA strand.

A measure of the average number of nucleotides that a template-dependent polymerase incorporates into a growing RNA or DNA chain before dissociating from the template DNA. Regulation of the processivity of a polymerase might involve interactions of regulatory factors with its sliding-clamp domain.

The process of correcting a misincorporation event by cleaving a short oligonucleotide sequence that contains the misincorporated residue from the 3′ end of the elongating transcript.

A chemical process that results in the sequential removal of single nucleotides from the 3′ end of the nascent RNA. It represents the direct chemical reversal of the single-nucleotide-addition reaction.

A process that decreases termination efficiency at a given terminator by increasing the rate of transcription by the RNA polymerase or by stabilizing the elongation complex, or both.

A domain or protein ring that forms part of the DNA-replication complex. It holds (or 'clamps') the replication polymerase to the DNA template at the primer–template junction within the replication fork. In transcription, this function is carried out by the 'pincer' domain of the 'crab-claw' region of the RNA polymerase. These pincers clamp the dsDNA downstream of the moving transcription bubble and control the processivity of the transcription complex.

The competition that is seen between different reactions during a process that can proceed down several bifurcating reaction pathways, for which the outcome depends on the relative rate constants of the potentially competing reactions. Alteration (for example, by transcription-factor binding) of the relative heights of the free energy of activation barriers that lead into the various reaction pathways can change the outcome of the process.

FREE ENERGY OF ACTIVATION BARRIER

An idea derived from transition-state theory that defines the input of free energy that is needed to allow a given reaction to proceed.

Describes how the total energy (or free energy, in this case) that is available for a reaction is divided among the population of molecules or pathways that are available within the system.

ISOENERGETIC REACTION PROCESS

A process in which the beginning and ending states have equivalent energies (or free energies).

The diffusion-driven process through which the elongation complex — which comprises the transcription bubble, the RNA–DNA hybrid and the RNA polymerase — translocates backwards and forwards along (and through) the DNA genome.

The process by which RNA polymerase might become detached from the 3′ end of the elongating RNA, and is translocated 'backwards' (upstream) by diffusion along the nucleic-acid framework towards the promoter, in response to regulatory signals or events during transcription elongation.

An enzyme that catalyses the cleavage of a single residue from the end of an oligonucletide strand. The term 'exonuclease-like' indicates that the enzyme might remove more than one residue at a time from the end of the chain.

The addition of an incorrect (as defined by the complementary DNA template) nucleotide at the 3′ end of the nascent transcript.


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