REP sequences: Mediators of the environmental stress response?

ABSTRACT

Repetitive Extragenic Palindromic (REP) sequences are highly conserved, structured, 35- to 40-nt elements located at ∼500 positions around the Escherichia coli chromosome. They are found in intergenic regions and are transcribed together with their upstream genes. Although their stable stem-loop structures protect messages against exoribonuclease digestion, their primary function has remained unknown. Recently, we found that about half of all REP sequences have the potential to stall ribosomes immediately upstream of the termination codon, leading to endonucleolytic cleavage of the mRNA, and induction of the trans-translation process. As a consequence, the mRNA and almost completed protein are degraded, and protein production from the affected gene is down-regulated. The process is critically dependent on the location of the REP element, with an effect only if it is within 15 nt of the termination codon. Using nrdAB as a model, we found that its down-regulation is affected by RNA helicases. Elimination of 6 helicases lowered NrdA production further, whereas overexpression of any RNA helicase partially reversed the downregulation. UV stress completely reversed down-regulation of NrdA production. Analysis of genes containing a REP sequence within 15 nt of the termination codon revealed that most, if not all, are up-regulated by environmental stress, as are RNA helicases. Based on these findings, we propose that REP-dependent downregulation serves as a mechanism to allow a rapid response to environmental stresses whereby RNA helicases partially open the REP elements enabling ribosomes to complete translation immediately increasing protein production from the affected genes.

Introduction

Cells regulate gene expression in response to intracellular and extracellular signals leading to alterations in their RNA and protein complements.¹ In bacteria, regulation occurs at multiple levels, and utilizes a diverse variety of mechanisms that affect mRNA production, mRNA stability, protein synthesis and protein stability.² As a result, cells are able to rapidly and efficiently satisfy their immediate needs for specific amounts of individual proteins (or RNAs). At the translational level, cells adjust their rate of protein production to alterations in environmental conditions such as nutrient deprivation or change in growth phase, temperature shocks, toxins, antibiotics as well as other stresses.³ In addition, stresses within the cell, such as stalled ribosomes, which can arise due to mRNA truncation, mRNA structure or codon usage, also elicit responses that alter the translation apparatus and affect protein production.⁴

Many regulatory processes are known that directly impact protein synthesis in response to environmental cues. Among these are factors that modulate the availability of functional ribosomes, a mechanism particularly important for cells in stationary phase. Also active during stationary phase are toxins that can cleave mRNAs and aid in re-programming translation for that specific phase of growth. Nutrient limitation, another stress, leads to elevation of the alarmones, ppGpp and pppGpp, which directly affect translation by competing with GTP for translation factors, and also indirectly by down-regulating transcription of RNA components of the translation apparatus.⁵ Translation is also regulated by mRNA structural rearrangements. These may involve trans-acting protein factors or regulatory RNAs that alter the ability of ribosomes to bind messages or they may impact cis-acting riboswitches that respond to environmental stimuli and lead to opening or closing a ribosome binding site. Other mechanisms of translational regulation have been identified that target other translation components such as tRNAs or aminoacyl-tRNA synthetases. Based on just this very brief summary, it is clear that so many different translational regulatory mechanisms have already been described that one cannot be faulted for believing that if a regulatory process can even be envisioned, some bacterial cell probably already uses it.

REP-dependent translational regulation

Despite this wealth of regulatory mechanisms, it was still quite surprising when we, together with Dr. Kenneth Rudd, identified a previously unknown process that regulates protein production through the action of Repetitive Extragenic Palindromic (REP) sequences.⁶ REP sequences are structured chromosomal elements widespread among bacterial species. In Escherichia coli, REP sequences are highly conserved, 35- to 40-nt in length, and located at ∼500 positions around the chromosome in intergenic regions downstream of termination codons.⁷ REP sequences can occur singly, but more often occur in clusters. They are transcribed together with their upstream genes, and consequently, lead to stable stem-loop structures in the 3’ UTRs of many mRNAs. Although REP elements protect mRNAs against the action of 3’ to 5’ exoribonucleases, their primary role in mRNA has remained unclear. Based on our recent studies,⁶ we believe that as many as half of the REP sequences in E. coli have the potential to serve as translational regulators.

Due to their extensive secondary structures, turnover of REP sequences in mRNA is dependent on RNase R and polynucleotide phosphorylase (PNPase), the only 2 exoribonucleases able to digest through structured RNAs. In the absence of these 2 nucleases, REP-containing RNA fragments accumulate to high levels.⁷ Detailed examination of the action of RNase R on REP-containing mRNAs revealed that, like non-stop mRNAs,⁸ these messages stimulate the binding of RNase R to ribosomes.⁶ However, this stimulation is critically dependent on the distance between the termination codon and the REP sequence with a sharp cutoff between 15 and 16 nt for the 3 genes examined. Movement of the REP sequence to a distance 16 or more nt from the stop codon eliminated its ability to stimulate RNase R binding to ribosomes resulting in stabilization of the ribosome-bound mRNA, and leading to a >3-fold increase in translation from that message. On the other hand, when the same REP sequence was within 15 nt from the stop codon, protein production was ∼3-fold lower.⁶ These data suggested that the REP element acts as a barrier to ribosome movement causing ribosomes to stall just before the stop codon thereby preventing completion of translation. Interestingly, earlier toe-printing experiments had indicated that 15 nt are the number of residues protected by ribosomes.⁹

Further examination of this phenomenon revealed that the transient stalling of ribosome movement leads to endonucleolytic cleavage of the mRNA and subsequent induction of the trans-translation process.⁶ The role of trans-translation was shown directly by the use of a strain carrying a mutant tmRNA in which the truncated peptide is not degraded. In this strain, the amount of protein produced was the same whether the distance between the stop codon and the REP sequence was within 15 nt or was 16 or more nt; however, the protein that accumulated when the distance was within 15 nt was truncated, but was not degraded as usual. These data showed clearly that the decreased protein production when the distance was within 15 nt was due to protein degradation as part of the trans-translation process. Additional support for the involvement of trans-translation came from the finding that mRNA was not degraded in a mutant strain lacking RNase R, a known component of the trans-translation system. Sequence analysis of the ribosome-bound mRNA in this strain revealed that it had been cleaved 4 nt upstream of the stop codon, and furthermore, that the cut site moves in concert when the position of the REP sequence is moved.⁶ The identity of the endoribonuclease responsible for the cleavage is not yet known; nevertheless, the fact that cleavage occurs due to REP-dependent ribosome stalling, generating the equivalent of a non-stop mRNA, explains why trans-translation comes into play.

All of the data are consistent with the conclusion that a REP sequence within 15 nt of a termination codon acts as a barrier to ribosome movement thereby preventing the A site of the ribosome from accessing the stop codon to enable the termination process to proceed. As a consequence, the ribosome stalls prior to the termination codon, the mRNA is cleaved, and trans-translation is induced, ultimately leading to degradation of the mRNA and the truncated peptide, and down-regulation of protein production from the upstream gene. Cleavage of the mRNA occurs between the 2nd and 3rd nt of the codon sitting at the A site, in agreement with earlier findings of Hayes and Sauer.¹⁰ However, when the spacing between the REP sequence and the termination codon is 16 nt or greater, the ribosome is able to fully access the codon, termination proceeds normally, and protein production is no longer impaired.

How many genes in E. coli have the potential to be affected by such a regulatory process? Analysis of the 496 genes with downstream REP sequences indicated that 260, or 52%, contain a REP element that is within 15 nt of the upstream termination codon. Thus, this regulatory mechanism could affect translation of a large number of genes. However, initial analyses of these genes provided no obvious commonalities that would explain why this group of genes is regulated in this manner.

The first hint of how the regulatory process might function physiologically came from the observation that for the 3 genes examined, the block to translation is leaky. In each case, ∼30% of the encoded protein continues to be produced when compared to the amount made when the stop codon-REP sequence distance is 16 nt or more. This information suggests that about one in 3 ribosomes is able to move a few additional nucleotides into the REP sequence enabling it to access the termination codon and complete translation. These findings imply that the base of the REP sequence may partially open, at least a portion of the time. How this would occur led us to consider that RNA helicases might play a role in the regulatory process.

Using the nrdAB message as a model, we found that in a strain lacking 6 RNA helicases (provided by Dr. Chaitanya Jain), production of NrdA protein and its mRNA was reduced even further, from the usual 30% to close to 10%, strongly supporting the conclusion that partial opening of the REP sequence by an RNA helicase was indeed responsible for the limited protein production derived from this gene. Conversely, overexpression of any one of the RNA helicases stimulated NrdA production ∼2-fold, slightly below the level obtained when the spacing was 16 nt or more.⁶ All of these findings strongly suggested that RNA helicases play an important role in REP-dependent regulation of translation, and, moreover, that environmental conditions that lead to elevation of RNA helicases would likely reverse, even if only partially, the downregulation induced by a REP sequence.

That this reasoning is, in fact, correct was suggested by the effect of UV treatment on the expression of NrdA. DNA damage induced by UV was known to increase expression of the nrdAB genes,¹¹ and we found that such treatment prevented the down-regulation of nrdAB message and NrdA protein that normally occurs in a wild type strain elevating them to the level found when the REP sequence is moved 40 nt downstream of the stop codon. Moreover, the UV treatment completely eliminated trans-translation,⁶ explaining why NrdA levels increased. This occurred despite the fact that in nrdA the REP sequence is only 9 nt downstream of its termination codon, well within the range in which it should have stalled ribosomes and impacted NrdA production. On the other hand, UV treatment did not affect expression from 2 other genes, avtA and fsaA, indicating that the effect was specific, and implying that other specific environmental signals might reverse REP-dependent downregulation of these genes. These findings lead us to propose that REP-dependent down-regulation serves as a rapid response mechanism to deal with stress conditions, and that elevation of one or more RNA helicases in response to a stress partially opens specific REP sequences to enable a greater number of ribosomes to complete translation of the upstream gene thereby immediately increasing protein production (Fig. 1).

Figure 1.

A model to explain how REP sequences regulate the expression level of stress-responsive genes under stress conditions. When the distance between a stop codon and the downstream REP sequence is 15 nt or less, ribosome stalling occurs because the REP sequence prevents its further movement. Subsequently, an endonuclease cleaves the mRNA generating a non-stop message that initiates the process of trans-translation. RNase R degrades the upstream portion of the cleaved message, and the polypeptide tagged by tmRNA is removed by proteases allowing ribosomes to recycle. Upon stress, RNA helicases are elevated that partially open the REP sequence and result in completed translation of the upstream gene leading to increased protein production. When the spacing is greater than 15 nt, normal translation occurs.

The stress response and REP-dependent translational regulation

Organisms are continuously challenged by diverse stress conditions, and to survive, they have evolved elaborate mechanisms to rapidly respond to these various stresses. One key mechanism is the expression of stress-responsive proteins in an effective and timely manner to help in dealing with the consequences of each particular stress For example, the nrdAB message and its protein product, discussed above, were elevated ∼3-fold within 10 min after UV treatment⁶ to help with DNA repair; ushA, which encodes UDP sugar hydrolase, is rapidly upregulated upon treatment with antibiotics;¹² the expression level of nagE, which is involved in N-acetylglucosamine transport, increases dramatically after cold shock or entry into stationary phase;^13,14 and sdhB, encoding succinate dehydrogenase, is up-regulated by antibiotics, cold shock and hydrogen peroxide (Table 1).^13-15 In fact, essentially all messages containing a REP element within 15 nt of its upstream termination codon appear to be elevated by one or more stress conditions,^13-16 consistent with our suggestion that translational regulation by REP elements serves as a rapid facilitator of the stress response. This mechanism, as well as others, ultimately leads to stress tolerance.

Table 1.

Examples of stress conditions that elevate REP-containing messages and RNA helicases .

Stress condition	Elevated REP-containing message	Elevated RNA helicase
antibiotics	rutR (S3), sdhB (S3), ushA (S9)	CsdA, Lhr, SrmB
cold shock	nagE (S6), sdhB (S3)	DbpA, RhlB, RhlE
oxidative	mrr (S8), narP (S5), tilS (S2)	CsdA, Lhr
stationary phase	nagE (S6), rutR (S3)	DbpA, Lhr, SrmB
UV	mrr (S8), nrdAB (S9)	CsdA, RhlB, RhlE, SrmB

The information presented was obtained from the microarray data at PATRIC.^13-17 A selection of stress conditions were chosen, and some REP- containing messages and the RNA helicases elevated by each stress are shown. The spacing (S) between the termination codon of each gene and its downstream REP sequence is indicated.

Most interestingly, the same stress conditions that elevate genes regulated by REP sequences also increase the levels of RNA helicases.^13-16 As shown in Table 1, each of the 6 known RNA helicases can be upregulated by certain stress conditions. In fact, a single stress condition usually increases the amount of several RNA helicases, suggesting functional redundancy among the helicases. In addition, a single helicase can be elevated by multiple stress conditions suggesting that it affects genes in a range of stress response pathways. These findings are all consistent with our proposal that REP-dependent translational regulation plays an important role in the response of cells to environmental stress, and that the response is mediated by the action of RNA helicases that open the REP sequences and enable rapid elevation of the products of stress responsive genes (Fig. 1). These conclusions raise the obvious question of how specificity is achieved inasmuch as a single stress elevates multiple RNA helicases, and each helicase can affect many REP-containing messages. We do not yet have an answer to this important question, but since many of the same stress-responsive proteins are required to ameliorate different stresses, perhaps this does not represent a major problem.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by Grant GM16317 from the National Institutes of Health to M.P.D., and grants 31370779 from the National Natural Science Foundation of China and from the Taishan Scholar Construction Foundation of Shandong Province to W.L.