Environmental factors control the expression of RpoS at the level of transcription, translation, and proteolysis (20
). Thus far, relatively few of the molecular links between environmental stimuli and the steps leading to changes in RpoS expression have been explained. We have analyzed how temperature controls the level of RpoS in the cell via the small regulatory RNA, DsrA. DsrA is necessary for the temperature control of RpoS and stimulates its translation (30
). We have found two major components of temperature regulation of RpoS by DsrA: (i) temperature-sensitive synthesis of the RNA and (ii) temperature-sensitive degradation. The net effect is a 25- and 30-fold decrease in full-length DsrA at 37 and 42°C, respectively, compared to 25°C (Fig. ; summarized in Table ).
Temperature regulation of DsrA synthesis appears to be responsible for an important portion of the differences in DsrA accumulation, particularly as the temperature increases from 37 to 42°C. While two control promoters, λpL
, increase slightly in activity at 37 compared to 25°C, the dsrA
promoter fusions were about twofold less active at 37 than at 25°C (Fig. and Table ). There is a further 2.5- to 3.5-fold decrease in the interval from 37 to 42°C; overall, synthesis from this promoter at 42°C is only 17% of what it is at 25°C (Tables and ). The minimal promoter contained within dsrAp36
is responsible for the majority of this temperature regulation; a second regulatory element located between −165 and −205 of the dsrA
promoter contributes further (Table ). We note that λpL
has previously been described as a temperature-sensitive promoter (14
). The work on λpL
utilized fusions containing the W205 trp
fusion with its temperature-dependent terminator; we believe that the behavior of this terminator explains those observations.
What is responsible for the temperature regulation of the dsrA
promoter? Our inability to recreate temperature regulation in vitro leaves open the possibility that a trans
-acting factor might be necessary for sensing temperature. However, if so, it must interact with the minimal promoter, which exhibits significant temperature regulation (Table ). Replacing the conserved spacer sequence of AAAAAAATTG with TCTAGAATTG did not change promoter strength or temperature sensitivity, inconsistent with an essential binding site for a protein in this region of the promoter. We favor instead changes in the structure of the promoter itself, possibly mediated by more general changes in the cellular milieu. The high AT content of the spacer, although not essential, may contribute to poising the system to facilitate melting during open complex formation. We have observed that the spacer is exquisitely sensitive to changes in size. The addition or deletion of 1 nt in the spacer decreases the activity of the promoter drastically, even at low temperatures. Other properties of the minimal promoter may help the dsrA
promoter to compete more effectively for RNA polymerase at low temperatures. For instance, the high-AT spacer, combined with other sequences, might allow open complex formation at 25°C more effectively than for other promoters. At higher temperatures, these features would be less of an advantage, and the dsrA
promoter would compete less well. Whether these features act independently, by directly promoting interactions with RNA polymerase, or respond to global changes in DNA topology, for instance, or chemical modifications, we do not yet know. For instance, the osmE
promoter is activated by osmotic shock, apparently leading to changes in supercoiling and therefore changes in promoter activation (9
). Such a requirement for a change in topology might explain our inability to see in vitro temperature regulation in a purified system and is being further investigated.
Within the upstream regulatory region for dsrA is the promoter for a divergent gene of unknown function (yedP or b1955). Using a fusion for this promoter containing the whole region between yedP and dsrA, we found that the yedP promoter is induced upon the entry into stationary phase and that its activity is not controlled by temperature. The expression of yedPp::frΔ strictly depends on RpoS (data not shown). The presence of this RpoS-dependent gene suggested the possibility of coupling between the transcription of dsrA and yedP. However, the activity of dsrAp205 was not affected in an rpoS mutant background (rpoS::Tn10) during growth at 25, 37, or 42°C (data not shown), conditions where the divergent yedP promoter is no longer active. It remains possible that the activity of this promoter or of factors regulating it affects the dsrA promoter under some conditions. We note the twofold negative effect of the far-upstream region (believed to contain the promoter) found at 42°C. Whether this is ever important for dsrA and therefore RpoS regulation is unknown.
Two regions of the dsrA
promoter stimulate synthesis in vivo and in vitro, the region from −46 to the −36 box (Fig. ) which contains conserved motif II and the region from −64 to −165. The sequence of motif II (AATATTT) is close to that proposed for the distal part of an UP element known to interact with the carboxy-terminal part of the α subunit (α-CTD) of the RNA polymerase (AAA[A/T][A/T]T[A/T]TTTT) (12
). Mutations in motif II (AATATTT changed to AGTATAC) reduce the stimulating effect of this sequence. Although the position of the sequence for dsrA
is closer to the −35 box than usually found, the stimulatory activity of this region in vitro (Fig. ) is most consistent with its action as a UP element. We have not characterized further the less conserved activator element(s) within the region from −64 to −165. The distance between the minimal promoter and region −64/−165 suggests that it is likely that the flexible carboxy-terminal part of the α subunit of the RNA polymerase would also be involved in these interactions. Whether use of either of these activating signals changes with growth conditions is not known.
LeuO, a regulator in the LysR family, down-regulates DsrA synthesis when overexpressed (24
). We find that LeuO is acting in a conserved region of the dsrA
promoter (motif III, Fig. ). Conditions affecting either the synthesis or the activity of LeuO or another LysR family regulator may significantly perturb DsrA synthesis and therefore synthesis of RpoS. This regulation is entirely independent of temperature. Thus, assuming that this conserved site in the dsrA
promoter and the effect of LeuO overexpression reflect a physiologically relevant regulatory signal, at least one other regulatory signal for DsrA synthesis besides temperature must exist.
While temperature provides a sixfold difference in the activity of the DsrA promoter between 25 and 42°C, the steady-state level of DsrA at 42°C is 30-fold less than at 25°C. The differences in the stability of DsrA at low and high temperature must contribute significantly to these differences in amounts, reflected in turn in differences in DsrA-dependent stimulation of RpoS (Table ). Furthermore, we found two different forms of DsrA, a full-length molecule (F) and a truncated molecule (T), probably missing the first stem-loop; the relative amounts of F and T also vary with temperature (Fig. and Table ). In independent work, Sledjeski et al. measured a half-life for full-length DsrA of 6 to 30 min (depending on the method for measuring the RNA) at 30°C and also noted the truncated species (46
). Since sequences within SL-1 are essential for RpoS translation (30
), we expect only the F form to be active for stimulation of RpoS; the decay of this form is particularly sensitive to temperature (Table ). Therefore, the degradation of the RNA molecule and the regulation of its synthesis will both participate in enhancing the differences in the steady-state levels of DsrA at low and high temperature.
Our data are most consistent with the T form of DsrA arising through cleavage of the full-length molecule (see above). In addition, T is relatively less abundant when DsrA is overproduced from a plasmid compared to the chromosome (Fig. and Table ), suggesting that the cleavage of DsrA to the T form may be limiting. If so, it may happen very quickly, possibly even before transcription has finished, because we did not observe the full-length molecule chasing into the truncated form after rifampin treatment (Fig. ). A sequence consistent with the RNase E cleavage site, GAAUUU, is present in DsrA at the base of SL-2, where we predict the cleavage to occur (11
). This region will be unpaired as the rest of the second stem-loop of DsrA is synthesized; it is also predicted to be unpaired from in vitro studies of the DsrA structure (26
The ratio between the two forms of DsrA (F/T) varies with temperature, with relatively more T form at higher temperatures (Table ). Processing of DsrA to the T form (in addition to general degradation) should act to block the formation of the RpoS-stimulating RNA but would allow accumulation of a form which may well be active on other targets. DsrA has been shown elsewhere to negatively regulate HNS synthesis (27
). A deletion of the first stem-loop of DsrA, leading to the expression of a mutant form similar to the T form, is still able to regulate HNS while losing RpoS regulation (30
). Possibly, processing allows DsrA activity on targets such as HNS to be less affected by temperature than regulation of RpoS (or other targets dependent on the first stem-loop). Thus, while degradation of the intact form of DsrA contributes to its overall temperature sensitivity, the specific cleavage of DsrA may allow channeling of the molecule to different targets.
In addition to the contributions of temperature to synthesis, degradation, and processing of DsrA, it is possible that DsrA activity is itself regulated by temperature. At 37 or 42°C, full-size DsrA was detected (Fig. ), but the expression of rpoS
is not dependent on DsrA (Table ). This suggest either that levels of DsrA at 37°C are below the threshold needed to stimulate RpoS translation or that temperature controls the activity of the molecule, either directly (changing the secondary structure of DsrA) or indirectly (acting on a factor controlling the pairing of DsrA with RpoS mRNA). Evidence does exist that the activity of DsrA on RpoS expression can change dramatically due to environmental changes. It has long been known that an osmotic shock increases translation of RpoS (34
). This lab has recently demonstrated that this increase requires DsrA but does not need an increase in DsrA amount (29
). Therefore, osmotic shock allows more efficient use of DsrA to stimulate RpoS translation and leads to DsrA-dependent translation at 37°C, even though, under other conditions, DsrA is not needed at 37°C (Table ). Temperature could affect DsrA activity in a similar manner. We tried to address this issue by synthesizing DsrA from a foreign promoter, pBAD. While induction of RpoS by DsrA was independent of temperature for this promoter, a great deal more DsrA was synthesized from pBAD at 42 than at 25°C; this lack of correlation between DsrA amounts and DsrA activity is again consistent with some temperature regulation of DsrA activity (data not shown).
The requirement for small, trans
-acting RNAs such as DsrA to stimulate RpoS translation allows the sensing of a variety of physiological conditions to result in major changes in the cell's capacity to react to stress. Two components of the temperature sensor for RpoS translation have been identified: the dsrA
promoter and the degradation of DsrA. Changes in synthesis and overall degradation with environmental variations such as temperature will affect RpoS translation as well as other targets of DsrA. HNS has been shown to be a target, and a number of other targets have been proposed (i.e., argR
). In addition, changes in the efficiency of the processing, as we have demonstrated here, should differentially affect the balance between these targets.
We now know that DsrA is only one of at least three small RNAs affecting RpoS translation. The other two small RNAs, RprA and OxyS, are synthesized in response to other signals (3
), significantly increasing the number of signals that can affect RpoS availability. It is becoming apparent that this is a paradigm for a new and significant level of cellular regulation. Multifunctional RNAs such as DsrA are likely to exist in all organisms. They allow integration of multiple environmental signals to coordinately regulate multiple outputs. Such a coordination can be added to independently operating transcriptional, translational, and posttranslational controls, providing even more responsiveness for the cell.