It should be noted that even shortening an sgrS variant from eight Ts to six Ts generated less functional SgrS, implying that six Us is not long enough to produce a fully functional sRNA Otaka et al. Because, from a structural perspective, the six U tail seems to be sufficient for binding to Hfq, a question remains what the role of extra U s plays in sRNA function in vivo. In addition, some sRNAs possess a discontinuous U-rich tail disrupted with other nucleotides Otaka et al.
Figure 1. The polyU tail of seven or longer is responsible for binding to the Hfq proximal face. B i : Termination at the seventh or longer position within the T stretch is necessary for functional sRNAs.
C Low temperature increases both the level of transcription initiation and the termination efficiency for the gene encoding the DsrA sRNA. D Termination efficiency at the sgrS terminator is improved by glucose-phosphate stress, which also induces transcription initiation of sgrS by SgrR, a transcriptional regulator.
Increased transcription termination will result in more production of SgrS and less expression of the downstream setA mRNA. In contrast to a conserved polyT stretch, nucleotide sequences for the stem-loop structure seem not to be conserved between intrinsic terminators Ishikawa et al. In fact, the SgrS sRNA was found to be fully functional with a heterologous terminator with a long polyU tail and hairpin stability approximately equal to that of the native terminator Otaka et al.
However, a dicF coding region expressed from a plasmid and lacking the downstream processing site still generated functional DicF in vivo Balasubramanian et al. Another important feature of the terminator stem-loop structure is the strength of the stem, which affects the position of termination. A moderate but not too strong stem is needed to generate a polyU tail of seven nucleotides or longer.
Extension of SgrS and RyhB terminator stems with additional G-C pairs resulted in premature termination and the generation of non-functional transcripts with polyU tails shorter than six Morita et al.
Based on the sRNAs studied thus far, the intrinsic terminators of sRNAs are expected to have unique features, forming a subset of the Rho-independent terminators in E. These features—a polyT stretch of seven or more and a moderate-strength stem-loop that enables termination after a stretch of seven or more Ts—can help in identifying potential Hfq-dependent sRNAs from genomic sequence analysis, and possibly help in distinguishing RNA transcripts that encode short proteins from those that have the potential to act as sRNAs.
This would also be useful for the design and engineering of synthetic sRNAs for the control of specific gene expression. The absence of the critical characteristics for Hfq binding in the intrinsic terminators for mRNAs might prevent these mRNAs from binding to and blocking the proximal face of Hfq, where sRNAs must bind.
SgrS is co-transcribed with the downstream gene setA encoding a SET sugar efflux transporter family protein, although SgrS requires transcription termination at its own terminator for function Sun and Vanderpool, ; Morita et al. Given that Rho-independent termination impacts functional sRNA production, the downstream genes are assumed to be expressed discordantly from these sRNAs. Although the biological significance of the discordant expression of sRNAs and the downstream genes remains to be determined, a recent study revealed that such discordant expression by internal Rho-independent termination occurs frequently within operons and contributes to preferred expression levels for individual proteins Lalanne et al.
Termination efficiency at the terminator for the DsrA sRNA, a positive regulator of RpoS translation, is increased at low temperature, where the dsrA promoter is most active Sledjeski et al. Similarly, an increase in termination efficiency at the SgrS and RyhB terminators was observed under conditions of both the cognate stress for transcriptional induction of these sRNAs and non-cognate stresses Morita et al. One can envision that this increase in termination efficiency would result in more effective production of sRNAs under specific conditions.
Previous studies on Rho-independent termination provide clues to the molecular mechanism by which Rho-independent termination is regulated. Lower levels of UTP nucleotide were found to improve transcription termination at several Rho-independent terminators in vitro Farnham et al. Therefore, this linkage between promoter strength transcription initiation and termination provides a way in which stresses that decrease overall transcription activity on a given gene may result in increases in the termination efficiency.
In addition, specific protein factors can be involved in regulation of Rho-independent termination. NusA is an essential protein which affects termination efficiency at Rho-independent terminators in multiple ways, by contributing to RNA polymerase pausing and by helping form and stabilizing RNA structures upon termination Nudler and Gottesman, ; Guo et al. Hfq can be excluded as a candidate factor because it seems not to be involved in the termination at sRNA terminators Morita et al.
Most in vivo experiments on Rho-independent termination have been carried out with protein-coding genes as templates. For instance, for short sRNA-encoding transcripts, dissociation of the sigma factor from the RNA polymerase, usually assumed to occur soon after transcription initiation, might not occur before the polymerase reaches the intrinsic terminator, possibly changing the efficiency of termination. A recent long-read RNA sequencing strategy enabled analysis of intact transcripts and revealed that the degree of readthrough of several mRNA terminators also varied between growth conditions Yan et al.
A critical question for future investigation will be how Rho-independent terminators are modulated and whether these mechanisms of modulation are specific to sRNA terminators, or also affect those for mRNAs. Thus, molecular or cellular processes that change ribosome occupancy on mRNA or the single-stranded nature of an RNA stretch at or adjacent to the rut site may regulate Rho's function.
Rho forms a homohexamer ring, and uses its N-terminal OB-like protein fold to bind cytosine-rich sequences, while a region near the C-terminal part of the protein binds to the RNA threaded in the channel; the latter binding event activates Rho's ATPase activity, driving its translocation Skordalakes and Berger, Several models of Rho-dependent transcription termination have been proposed Peters et al.
In vitro approaches to study Rho-dependent termination and its regulation by sRNAs have been recently described Nadiras et al. In vivo approaches primarily depend on defining Rho-dependent termination with Rho-specific inhibitors such as bicyclomycin, and evaluating effects of sRNAs on mRNA expression by measuring read-through products with reporter assays, quantitative PCR or deep sequencing Hussein et al. Rho can also terminate transcription inside genes and within operons, and this is likely a highly regulated process given the fact that the translation status of mRNAs can directly affect Rho access to rut sites inside coding sequences Adhya and Gottesman, The observation of such intragenic termination sites raised the possibility that factors that can affect translation, such as sRNAs or RNA-binding proteins, may regulate Rho-dependent termination at these sites.
Interestingly, ChiX-mediated translation inhibition has a secondary effect. By blocking entry of ribosomes on chiP mRNA, ChiX binding exposes an intragenic rut site for Rho loading, leading to premature termination of transcription inside the chiP coding sequence Figure 2A.
This regulation not only reinforces the inhibition of chiP translation but also leads to a polarity effect, inhibiting expression of the downstream chiQ gene in the same operon Bossi et al.
When chitosugars are available and thus the transporter is needed, ChiX is destroyed by interaction with an RNA decoy, induced dependent on chitosugar sensing Figueroa-Bossi et al.
Therefore, regulation coordinates with other transcriptional circuits to ensure genes responsible for utilization of chitin-derived oligosaccharides are switched on only when chitosugars are present.
In a second example, the E. This type of regulation allows discoordinate expression of operon genes in response to metabolic needs of the cells. Figure 2. When the accumulation of Salmonella ChiX or E. When ChiX or Spot42 sRNA is produced at high levels, under specific environmental conditions, sRNA annealing blocks the ribosome binding site of the corresponding mRNA target chiP or galK , respectively ; reduced translation allows Rho loading onto a cryptic rut site to terminate transcription inside the genes dotted pale portion of box represents untranscribed portion of the mRNA.
In the cases described above, sRNAs facilitate Rho access to rut sites by interfering with mRNA translation, consistent with the consensus that mRNA sequences devoid of translating ribosomes are preferable targets for Rho binding and regulation.
However, sRNAs can also positively regulate genes, raising the possibility that in some cases, sRNAs might antagonize Rho function by blocking Rho binding and termination.
Transcriptomic analyses of RNA samples from E. Such a regulatory mechanism is likely not limited to rpoS , but may apply to many other putative targets of these three sRNAs, as well as other positively regulated mRNAs. As optimal growth of E. While the physiological role of this regulatory interaction is not known, it raises the possibility that under certain conditions the cell might modulate Rho levels and thus Rho-dependent termination via the SraL sRNA.
For both the rpoS and rho leader, it is still not clear how sRNA binding prevents Rho-dependent premature termination. Therefore, it is very likely that sRNA blocks Rho loading by occluding a rut site, either directly by overlapping the site, or indirectly via RNA structure rearrangements. The latter regulatory mechanism is exploited by the E.
Rabhi et al. Nonetheless, these reports of both negative and positive regulation of Rho-dependent termination by Hfq-dependent sRNAs or RNA-binding proteins reinforce the idea that RNA-based regulation of termination is widely exploited by bacteria.
Rho is an abundant protein in many bacteria, including E. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We surmise that Rho might compete with, or augment, the Mfd function. In bacteria, the transcription process ends via two types of termination pathways, namely, intrinsic or factor-independent and Rho-dependent termination 1.
Eventually it catches up the elongation complex EC and dissociates it 2 , 3. Transcription elongation factor, NusG, interacts with Rho and stimulates the termination process 2 , 3. Recent studies have revealed that by the virtue of its RNA-binding property and due to the all-pervading nature of the Rho-dependent termination, the Rho protein is found to be involved in the various physiological processes 3 , 4 , 5. During the bacterial growth, its DNA can be damaged by several extraneous physical radiations or chemical factors hazardous chemicals or metabolites.
To cope up with these stresses and to maintain the integrity of the DNA, bacteria have evolved the base excision repair BER system to remove single base aberrations and the nucleotide excision repair NER to remove large DNA lesions and the abasic sites 6 , 7 , 8 , 9 , UvrD, a helicase, unwinds the excised fragment and removes it. A whole body of literature has provided evidence that Mfd plays a pivotal role in initiation of the TCR, especially in repairing the damages of the transcribing strand 6 , 15 , 16 , 17 , However, in an alternative model, it has been claimed that a Mfd-independent pathway is also operational to dislodge the RNAP, where UvrD together with NusA induce the backtracking of the stalled ECs so that the DNA lesions are exposed to the other Uvr proteins 19 , The transcription elongation is a discontinuous process.
Depending on the DNA sequence and structures, the transcription elongation slows down or the RNAP stalls at the specific pause sites, which has various physiological consequences Stalled EC, not only masks the damaged sites from the DNA repair but also could cause general hindrance to several DNA-dependent processes, which in turn would cause lethality.
So, it is likely that a significant fraction of the ECs will be the target of the Rho, especially those engaged in the non-operonic transcriptions. Consistent with that idea, recently it has been shown that one of the major roles of Rho is to prevent the pervasive transcription Earlier, we have shown that Rho can dislodge stalled ECs in vitro Hence, we hypothesized that Rho, by inducing transcription termination, is capable of recycling the RNAP stalled at the DNA lesions and thereby, might facilitate the repair of the damaged sites by unmasking them to the DNA repair machineries, and in this regard, it resembles the RNAP displacement activity of the Mfd Fig.
Hypothesis of connecting Rho-dependent termination and NER : Cartoons depicting a operonic transcription starting from the promotor region of a gene and pervasive transcription starting from other cryptic start sites transcription.
Here, we show that Rho mutants and NusG mutants NusG is a transcription elongation factor that interacts with Rho and stimulates the termination process exhibit synthetic growth defects with mfd. The E. Failure of this recycling leads to reduced survival of E. We propose that Rho might either compete or augment the function of Mfd in vivo. If Rho-dependent transcription termination process is involved in dislodging the ECs stalled at the DNA lesions and recycling the RNAPs, then in the presence of Rho mutants, defective for the termination function, should affect the DNA-dependent processes like DNA repair and the strains having these mutants should exhibit higher sensitivity towards the DNA-damaging agents.
Also, failure of the RNAP-recycling is likely to lead to the reduction of survival of the cells. Hence, we explored to exhibit these in vivo phenotypes of the strains having the Rho mutants. The synthetic growth defect in bacteria occurs when simultaneous perturbation of the two genes results in lethality or drastic reduction in the rate of growth. Determination of synthetic defects is a very useful tool to establish the genetic interactions between the two gene products.
In the WT E. We employed synthetic growth defect assays by deleting each of the NER genes uvrA, uvrB, uvrC, uvrD and mfd; see Methods section for the deletion procedures individually from the E. These Rho mutants are defective in ATPase as well as termination activities 30 , whereas the NusG mutants are defective for the Rho-binding We observed the following.
II When mfd was deleted from the Rho mutants, the growth rate was reduced significantly Fig. III When the strains were expressing the NusG mutants, the synthetic defects were less severe, in all the cases, except that for the mfd data not shown.
These synthetic effects were specific to the NER genes as the deletion of an unrelated gene, uhpt , did not elicit similar response supplementary figure 2b. Synthetic growth defects of the Rho mutants with the different NER genes. Error bars were calculated from the standard deviations obtained from at least three independent measurements. Source data are provided as a Source Data file. These results indicated that Rho-dependent termination might be involved in dislodging the ECs stalled at the lesions and the process is genetically connected to the Mfd function.
As Mfd function becomes most essential when Rho-dependent termination process is compromised, both of them might function in synergy. As the dislodging of the EC and the unmasking of the DNA lesions is essential for the initiation of the repair process, it is likely that the E. These agents create covalent adducts to the DNA that blocks the progression of the transcription EC 22 , Different derivatives of the MG strains see Fig.
Next, we monitored the growth of different strains in the absence and presence of Cisplatin. Sensitivity of E. Rho mutants were more sensitive towards Mitomycin C 0. Rho mutants and various uvr deletion strains showed high sensitivity towards cisplatin compared to the WT strain. The plasmid pBR exposed to the increasing doses of UV was transformed to the strains either expressing mutant rho or are deleted for either uvrA or mfd.
Fractions of number of transformants obtained were plotted against the UV-dose used to damage the plasmid in vitro. If the NER pathway is impaired, the E. To further confirm the involvement of Rho-dependent termination in removing these stalled complexes, we monitored the UV-dose sensitivity of the strains expressing the Rho mutants, as well as the strains having deletions in the uvrA, uvrD , and mfd.
In an old study, a Rho mutant, rho15 , bearing strain was observed to be UV-sensitive, but the phenomenon was not linked to the NER process Next, we measured the in vivo DNA repair proficiencies of the aforementioned strains expressing Rho mutants and compared them with those having uvrA or mfd deletions, which is a direct measurement of the involvement of Rho-dependent termination in invoking and facilitating the NER pathway Fig.
A plasmid DNA was damaged by exposing to the increasing doses of UV, and the in vivo DNA repair ability of the different strains were assessed by counting the number of transformants on the plates formed after the transformation with this damaged plasmid. However, we cannot rule out the possibility that the Rho mutants affected the DNA propagation step s instead of the DNA repair step s , because the former would also have a similar manifestations of poor survival of the transformants.
The aforementioned several lines of evidences also suggest that the severity of phenotypes induced by the Rho-mutants is comparable to those observed when the strains are devoid of the Mfd protein. To further provide evidences that the Rho-dependent termination and the Mfd functions belong to the same pathway s of the NER process, we wanted to observe the combinatorial effect on the NER when both these functions are compromised simultaneously.
In Fig. Hence, we used an E. The growth of these resultant strains were monitored after deleting the chromosomal mfd Fig. Combinatorial effects of the Rho mutants and either the Mfd deletion or the Mfd mutant. In combination with rho Y80C, the deletion of mfd , induces severe slowing down of the bacterial growth.
Error bars were calculated from the standard deviation obtained from at least three independent measurements. Aforementioned strains expressing different variants of the Rho in the presence and absence of the Mfd protein were subjected to various UV doses Fig. The UV-sensitivities were milder when either Rho or Mfd functions were separately disabled or were functioning less efficiently see also Fig.
This additive effect, when both the Rho and Mfd functions are compromised, on the UV-sensitivity, as well as on the cell growth further suggest that the presence of one of them in the cell is essential for the invocation of the NER as well as the survival of the cells. Rho is capable of only displacing the stalled EC If the Mfd and the Rho function in synergy, a constitutively acting Mfd mutant capable of functioning without interacting with RNAP is likely to suppress the synthetic growth defects caused by the Rho mutants and the mfd deletion.
As was expected, we observed that this mutant did not exhibit synthetic growth defect with the Y80C Rho mutant Fig. We confirmed the existence of these adducts by the sensitivity of this damaged template to an enzyme called, T4-PDG from bacteriophage T4, pyrimidine dimer glycosylase; NEB Fig.
We then proceeded with the transcription reactions on this template, both in the presence and absence of either Rho or Mfd proteins Fig. In vitro transcription assays to measure RNA release. Promoter and the terminator regions are indicated. ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site.
Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60—90 nt.
Rho and Mfd proteins are indicated. Hairpin structure ceases the RNA polymerase activity. Moreover, in the U rich regions, there are weak interactions between U bases of transcript and A bases of the template. In the end, transcript liberates from the transcription site. Rho dependent termination is mediated by a Rho protein while Rho independent termination occurs via the formation of a hairpin loop structure. So, this is the key difference between Rho dependent and Rho independent termination.
Therefore, this is also a significant difference between Rho dependent and Rho independent termination. The below infographic shows more comparisons related to the difference between Rho dependent and Rho independent termination.
Our results demonstrated that RhoTermPredict is a very performing algorithm both for limited-length sequences F 1 -score obtained about 0. Furthermore the degree of overlap with ARNold predictions was very low. Our analysis shows that RhoTermPredict is a powerful tool for Rho-dependent terminators search in the three analyzed genomes and could fill this gap in computational genomics.
We conclude that RhoTermPredict could be used in combination with an intrinsic terminators predictor in order to predict all the transcription terminators in bacterial genomes. Intrinsic terminators are characterized by an RNA structure having a GC-rich hairpin immediately followed by a stretch of 6—8 uridine residues [ 1 , 2 ], while Rho-dependent terminators rely upon the interaction of a protein called Rho with the RNA Polymerase RNAP [ 1 , 3 , 4 , 5 ].
This site is known as the Rho utilization site the so-called RUT site. The depletion of G within a natural RUT site minimizes the formation of potentially interfering secondary structures, which generally inhibit Rho binding [ 1 , 8 , 9 , 10 ]. According to a widely accepted model, Rho catches up to the elongation complex by translocating along the nascent transcript and, at certain pausing sites, dissolves the elongation complex by pulling out the transcript [ 1 , 3 , 4 , 6 , 11 ].
Allosteric interactions between Rho and RNAP facilitating catalytic inactivation and eventual dissociation of the elongation complex have been also more recently proposed [ 11 , 12 ].
Over the past decade, a lot of studies, performed in several bacterial species, have established the importance of Rho in gene regulation and its conserved role in the enforcement of transcription-translation coupling, by interrupting transcription of untranslated mRNAs [ 14 , 15 , 16 ]. Furthermore, in Escherichia coli , Bacillus subtilis , Staphylococcus aureus , and Mycobacterium tuberculosis an important role of Rho in suppression of pervasive, primarily antisense transcription was demonstrated [ 17 , 18 , 19 , 20 ].
Complete or even partial inactivation of Rho in these bacterial species causes widespread transcription originating from cryptic promoters and read-through of transcription terminators [ 21 ].
Conversely, Rho-dependent terminators have so far proved difficult to predict computationally because the sequence features required for the function of Rho are complex and poorly defined, in contrast to intrinsic terminators features [ 1 ]. Hence at the moment, other Rho-dependent terminators prediction programs that take into account all the steps of the Rho-dependent transcription termination have not yet been created. Rho is very often present in bacterial genomes and the basic principles of Rho-dependent-termination are conserved across species, despite some structural differences between Rho proteins [ 21 ].
For this reason the implementation of an algorithm for the prediction of terminators mediated by Rho factor could be certainly very useful. In this study we introduce RhoTermPredict, a novel algorithm for the prediction of transcriptional Rho-dependent terminators in E.
RhoTermPredict is the first program implemented for an exhaustive search of Rho-dependent terminators, which functions in two steps to specifically identify this type of transcription termination sites within a genome sequence. Our aim was to create a program for the prediction of such elements in a prokaryotic genome based on a conserved structured motifs search, in a similar way to our previous work regarding the promoter prediction algorithm G4PromFinder [ 27 ].
We tested RhoTermPredict performances by using available genomic and transcriptomic data of the model microorganism E. In order to estimate the degree of overlap between Rho-dependent and intrinsic terminators predictions, we also run one of the currently available tools for bacterial intrinsic terminators prediction on the same sets of E.
In addition to E. RhoTermPredict algorithm was written in the Python v. It accepts as input bacterial genome sequences, and provides as output the coordinates of putative Rho-dependent terminators elements RUT and RNAP pause sites with a score assigned to them that indicates the probability that the extracted region actually corresponds to a Rho-dependent terminator see below for the scoring assignment method.
For Rho-dependent terminator predictions, we used available genomic sequences of the model microorganism E. For the prediction quality evaluation, we used E. In fact, although the transcription termination mechanisms in B. The procedure to search for putative Rho-dependent terminators is reported below. RhoTermPredict actually searches for some mandatory elements putative RUT and RNAP pause sites and for other optional elements whose presence could increase the prediction score.
In fact, a consensus motif common to all Rho-dependent transcription terminators has been previously proposed in E. On that basis, we elaborated the following two-step procedure to detect Rho-dependent terminators Fig. Method used for the prediction of putative Rho-dependent terminators.
RhoTermPredict also allows to predict multiple putative terminators in a single query region, and to search for terminators in both strands.
The maximum score that could be assigned by our algorithm to a terminator prediction is 15, while the minimum is 6 a minimum of 3 point for the RUT site and also a minimum of 3 points for a pause site. Therefore a differential expression of these BSTs regions indicates the presence of a Rho-dependent transcription terminator. Starting from the genome of E.
The positive set consisted of sequences. The negative set consisted of sequences. For B. As for E. We considered as either true positive TP or false positive FP any sequences of either the positive or the negative set in which the algorithm predicted a terminator, respectively.
Importantly, at most one TP was considered for each sequence of the positive set. We considered as either true negative TN or false negative FN any sequences of either the negative or the positive set in which the algorithm did not predict any terminator, respectively.
Alignment to the reference strain of E. Evaluation of strand specificity and gene coverage was performed using BEDTools v2. To avoid bias caused by multi-mapping reads the non-deterministic option and end-to-end mode were used to force a single assignment of multi-mapping reads to the best scoring region if present or in the case of regions with identical scores reads were randomly assigned.
Mapped reads with MAPQ mapping quality greater than 30 were analyzed to determine the read counts per protein-coding gene. We also run RhoTermPredict on the whole genome of E.
To validate these predictions, we used the RNA-Seq data above described. We considered the predicted regions as putative Rho-dependent terminators if they were in positions in which there was a negative transcription gradient in RNA-Seq data, as suggested in other works [ 17 , 21 ].
Precisely, we considered, in this case, a prediction as TP if there was a decrease of read value by a factor of at least 1. In this study we used as data sources: i. The complete genome of E. The results obtained are shown below. Moreover, we decided to test RhoTermPredict also on genome sequences of B. Table 1 summarizes statistics of putative Rho-dependent terminators that were predicted by RhoTermPredict in the positive set.
All predicted terminators without any limitation on the prediction score were taken into account. In the positive set of sequences, the algorithm predicted putative terminators in most A total of putative terminators were predicted. Multiple putative terminators were sometime associated with single sequences of the positive set.
In particular, in
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