fujix p401 manual

A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These ?ndings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. The results indicated not only the primary importance of base pairings between the translation initiation region of ca. 20 nucleotides (the AUG initiation codon plus the “downstream box”) and the internal region of rpoH mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift. The heat shock response is a universal, adaptive, and ho- meostatic cellular response against damage to protein folding under heat and other stresses. In Escherichia coli, the response results primarily from a transient increase in the level of s 32, which is encoded by rpoH and which is speci?cally required for the transcription of the set of well-conserved heat shock genes (11, 35). The increase in the s 32 level results from both the enhanced synthesis and the stabilization of normally unstable s 32 (12, 30). The production of abnormal proteins under various con- ditions also induces the heat shock response through an in- crease in the s 32 level (10), but such induction appears to involve only the stabilization and not the increased synthesis of s 32 (16). As to the mechanism of translational induction of s 32, ex- tensive deletion analyses of an rpoH-lacZ gene fusion revealed the involvement of positive and negative regulatory regions (regions A and B, respectively) on the 5 9 portion of rpoH mRNA (15, 21). Region B, an internal coding segment of ca.

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You are currently using an outdated browser. To optimise your browsing experience, please update your browser. You can update your preferences, withdraw your consent at any time, and see a detailed description of the types of cookies we and our partners use in our Cookie Policy. By clicking on an item on our website, you agree to our use of cookies. Learn more about our cookies and your options. A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. Schematic representation of the 5 portion (nt 19 to 247) of E. coli rpoH mRNA as predicted by use of Mulfold (14). (A) Secondary structure thought to be involved in modulating heat-induced synthesis of 32 (21). Region A (nt 6 to 20), the initiation codon, and the Shine-Dalgarno (SD) sequence are indicated. Region B (nt 112 to 208) is shaded. The locations of regions A and B are indicated. (B) SDS-PAGE patterns of fusion proteins expressed from the wild-type and mutant forms of TLF247. Structures predicted for RNA of 150 nt (starting from nt 19 for each; the sequences may include a BamHI junction and part of lacZ) and that have minimum free energy are shown for some representative constructs examined in Fig. 4. Only relevant portions are presented. The initiation codon, region A, and region B are indicated as described in the legend to Fig. 3B. The Shine-Dalgarno sequence is shown by shaded letters.We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes.

rst probed the structures of rpoH RNAs from the wild type and several mutants in vitro and then analyzed their expression in vivo after transcription from a single-copy rpoH-lacZ gene fusion. The data supported some salient features of the pre- dicted mRNA secondary structure and provided the basis for further analysis of each of the component stem-loop struc- tures. The results led us to propose that an mRNA secondary structure with appropriate stability and formed between the translation initiation region (the AUG initiation codon and region A) and the internal coding region is a prerequisite for the thermoregulation of s 32 synthesis. The l TLF97-3 vector (28) was used to construct rpoH-lacZ gene fusions. Recombinant DNA and other general techniques were as described by Sambrook et al. (26) and by Miller (20). Chemicals, enzymes, and buf fers. 1-Cyclohexyl-3-(2-morpholinoethyl)-carbo- diimide metho- p -toluene sulfonate (CMCT) and diethyl pyrocarbonate (DEP) were purchased from Sigma. RNase V 1 was obtained from Pharmacia, and avian myeloblastosis virus reverse transcriptase was obtained from Life Science. Buf fer H was 70 mM HEPES-KOH (pH 7.8) containing 10 mM MgCl 2, 270 mM KCl, and 1 mM dithiothreitol, and buf fer V1 was 30 mM Tris-HCl (pH 7.8) containing 20 mM MgCl 2, 300 mM KCl, and 1 mM dithiothreitol. Construction of rpoH-lacZ gene fusions. The gene fusion (translational fusion) designated TLF247 was constructed by in-frame fusion between the Xho I- Bam HI fragment of pGF247 (21) containing the rpoH promoters and the 5 9 portion of the coding region (nt 2 677 to 1 247) and codon 9 of lacZ on the l TLF97-3 vector. The same fragment of pGF247 was also inserted into pBlue- script SK( 1 ), yielding pBSK247. Derivatives of TLF247 carrying base substitu- tions were constructed by PCR with plasmid pFRP103 containing each of the FIG. 1. Schematic representation of the 5 9 portion (nt 2 19 to 1 247) of E. coli rpoH mRNA as predicted by use of Mulfold (14).

100 nt, is a negative element involved in re- pressing translation under nonstress conditions. A computer prediction revealed a secondary structure for the 5 9 segment (nt 2 19 to 1 247) of rpoH mRNA which is fully consistent with the above ?ndings; base pairings between region A and part of region B appeared to negatively modulate rpoH translation (21) (Fig. 1). Mutational analyses of rpoH mRNA de?cient in the expres- sion or regulation of a GF364 fusion carrying the initial 364 nt of the rpoH coding region (Fig. 2A) not only substantiated the importance of some of the critical base pairings but also sug- gested the possible involvement of speci?c nucleotide se- quences in heat induction (36). It was surmised that the trans- lation of rpoH mRNA is restricted by the formation of secondary structure(s) that would limit ribosome entry under nonstress conditions. In addition, the isolation and characterization of rpoH homologs from a number of gram- negative bacteria revealed evolutionary conservation of both region A and the mRNA secondary structure among the gamma proteobacteria (23). All members of the latter group of bacteria examined seemed to exhibit heat-induced synthesis of s 32 homologs at the translational level, as in E. coli (24). The translational induction of s 32 is transient and is fol- lowed by a shutof f phase mediated by the DnaK-DnaJ-GrpE chaperones (9, 12, 29). The translational repression and desta- bilization of s 32 during adaptation periods are part of the feedback regulatory mechanisms (29, 31, 32) mediated by a segment of s 32 protein (18, 22, 35) which contains a highly and uniquely conserved sequence among the rpoH homologs (23, 34). Mailing address: HSP Research Institute, Kyoto Research Park, Kyoto 600-8813, Japan. Phone: (81)-75-315- 8619. Fax: (81)-75-315-8659.We ?

ed with a Fujix BAS2000 imaging analyzer to determine the rates of synthesis of fusion proteins after correction for recovery with v protein as a reference. RNA preparation. RNA containing the upstream region and part of the rpoH coding region (nt 2 60 to 1 247) was prepared in vitro with T7 RNA polymerase by use of an RNA transcription kit (Stratagene). The A? II- Bam HI fragments of pBSK247 were placed under the control of the T7 promoter of vector pSP72, and the resulting plasmids were digested with Bam HI and used as templates for RNA synthesis. Structure probing of RNA. The procedures used for RNA structure probing were essentially those described by Christiansen et al. (3). Prior to treatment with CMCT, DEP, or RNase V 1, RNA (4 m g) was renatured (heating and slow cooling) in 20 m l of buf fer H, 200 m l of buf fer H, or 20 m l of buf fer V1, respectively. RNA was treated with CMCT (50 mM) or DEP (96 m M), and the reaction was terminated by the addition of ethanol on dry ice. For RNase V 1 treatment, 6 m l of RNA was mixed with an equal volume of buf fer V1 containing enzyme on ice for 30 min, treated with phenol, and precipitated with ethanol. RNA incubated without probes served as a control in all experiments. The identi?cation of modi?ed bases was carried out by primer extension analysis: 0.3 pmol of modi?ed RNA and 3 pmol of 5 9 -?uorescence-labeled primer comple- mentary to the 5 9 (nt 1 79 to 101) or 3 9 (nt 1 227 to 247) region were incubated with avian myeloblastosis virus reverse transcriptase. RESULTS Thermoregulation of a TLF247 gene fusion mediated by an mRNA secondary structure. To further understand the rpoH translational control mechanisms, it was important to analyze structural features of the “minimal” mRNA segment(s) essen- tial for thermoregulation. The locations of regions A and B are indicated. (B) SDS-PAGE patterns of fusion proteins expressed from the wild-type and mutant forms of TLF247.

(A) Secondary structure thought to be involved in modulating heat-induced synthesis of s 32 (21). Region A (nt 1 6 to 20), the initiation codon, and the Shine-Dalgarno (SD) sequence are indicated. Region B (nt 1 112 to 208) is shaded. Numbers refer to the nucleotides of the coding sequence. (B) Putative base pairing between the downstream box (region A) of rpoH and the “anti-downstream box” of 16S rRNA (spanning nt 1469 to 1483). F, G-U pairs. 402 MORITA ET AL. J. B ACTERIOL. Seven extra bases containing the Bam HI site were added to the latter primer to make in-frame fusions to lacZ (21). A set of 3 9 deletions of GFR153 was constructed by PCR with the same 5 9 primer as that used above and 3 9 primers that corresponded to the end of each deletion (with the same seven extra bases) and with l GFR153 (21) as a template. DNA fragments with the desired sequences containing PCR-ampli?ed products were inserted into pBSK247, and nucleotide sequences were con?rmed by dideoxy sequencing. The Xho I- Bam HI fragments of the resulting plasmids were then transferred to the l TLF97-3 vector by in vitro packaging. TLF229 D (stemIII) was constructed from TLF247 by de- leting the apical portion of stem II (nt 1 30 to 110) and all of stem III (nt 1 128 to 178). Four synthetic oligonucleotides (ca. 60 nt long) were annealed and ligated to create a DNA fragment with 5 9 protruding ends for joining with the Cla Io r Bam HI site at the 5 9 or 3 9 end, respectively. The resulting fragment was cloned into pBSK247 by replacing the Cla I- Bam HI fragment to obtain pBSK229 D (stemIII). Determination of rates of synthesis of fusion proteins. The procedure used for the determination of fusion protein synthesis rates was essentially that described previously (21). The immunopre- cipitates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (7.5% gel), and the intensities of radioactive bands were quanti?

3B probably represents a major but not the only structure found under the set of conditions used. Altered secondary structure of some mutant rpoH RNAs. To determine the ef fects of base changes on the mRNA secondary structure, RNAs prepared from ?ve mutants examined above (Fig. 2B) were subjected to similar structural analyses (Fig. 3A). The results obtained, combined with those for the wild- type RNA, revealed certain interesting dif ferences as well as similarities (Table 1). Evidently, stems II and III in the 15A and 15C mutant RNAs were modi?ed to greater extents than those in the wild-type RNA. A-15 was clearly modi?ed by DEP in 15A RNA; in contrast, G-15 in wild-type RNA was not modi?ed by CMCT as expected. More importantly, U-14 was also modi?ed strongly in 15A RNA but not in wild-type RNA, indicating that the neighboring structure was af fected by the G-to-A mutation at 1 15. Since C should not be modi?ed by either probe, the change at 1 15 could not be seen in 15C RNA. However, U-14 was not modi?ed in 15C RNA, indicat- ing that the change in the neighboring structure was more pronounced in 15A RNA than in 15C RNA. Further deletion analysis of critical regulatory regions. To further de?ne the rpoH regions critical for thermoregulation, we constructed and examined a set of 5 9 and 3 9 deletions of the rpoH-lacZ fusion on l TLF247. These results also suggested that the apical portion of stem II and the intact form of stem III were not essential for regulation.These results appeared to indicate the impor- tance of stem I but not stem IV for thermoregulation within the limitations of these experiments. In contrast, the apical portion contains A-18 to C-26, A-154 to C-172, and seven extra bases inserted during construction (Fig. 5B, broken line) and dif fers drastically from that of the parental TLF247 fusion (Fig. 3B). Thus, although the basal portion around the translation initiation region appeared to be most important, it was not suf ?

cient for ef fective thermoregulation. A minimal gene fusion that can respond to heat shock. Segments of mRNA that correspond to each of the stem structures (I to IV) are shown above the diagram, and nucleotide numbers are shown below. Regions A and B are indicated by stippled and hatched boxes, respectively. (B) A pair of deletions lacking stem III.These results, combined with the RNA secondary structure prediction for various constructs, sug- gested that the mRNA secondary structure involving the trans- lation initiation region (the initiation codon plus region A) with appropriate stability or instability may be a primary re- quirement for the thermoregulation of rpoH translation. DISCUSSION The ef ?ciency of translation in E. coli is determined primar- ily at the stage of initiation, which includes binding of the 30S ribosome to 5 9 segments (from approximately nt 2 20 to 1 15) FIG. 5. Predicted mRNA secondary structures for some of the deletion derivatives used. Structures predicted for RNA of 150 nt (starting from nt 2 19 for each; the sequences may include a Bam HI junction and part of lacZ ) and that have minimum free energy are shown for some representative constructs examined in Fig. 4. Only relevant portions are presented. The initiation codon, region A, and region B are indicated as described in the legend to Fig. 3B. The Shine-Dalgarno sequence is shown by shaded letters. Arrowheads indicate the positions where two G’s were replaced in constructing TLF229 D (stemIII)GG (Fig. 4B). The broken line indicates extra bases inserted during construction (21). 408 MORITA ET AL. J. B ACTERIOL. Thus, the secondary structure of such mRNA segments can play an important role in modulating translation ef ?ciency (5). In the case of rpoH, part of the ribosome binding site (nt 1 1 to 20) including the AUG codon and region A (downstream box) was thought to be masked through the formation of base pairs with the internal region (region B).

Closed and open arrows indicate fusion proteins and b -galactosidase v protein (internal reference), respectively. V OL. 181, 1999 TRANSLATIONAL CONTROL OF s 32 SYNTHESIS 403 A 5 9 segment (nt 2 60 to 1 247) of wild-type (WT) or mutant RNAs prepared in vitro was treated with CMCT, DEP, or RNase V 1, and modi?ed bases were identi?ed by reverse transcription analysis as described in Materials and Methods. Only some of the bases that were clearly modi?ed by each treatment are indicated by nucleotide numbers. Relevant sequence ladders (wild type) are shown to the side as a reference. When comparing the observed bands with the sequence ladders, one should note that cDNA synthesis stops one residue before the modi?ed base. Arrowheads indicate some of the modi?cations uniquely found in mutant RNA(s), whereas arrows indicate positions at which reverse transcriptase was arrested. Only the data for wild-type RNA are shown for experiments with RNase V 1. (B) Modi?ed bases identi?ed with wild-type RNA in panel A are indicated on the mRNA secondary structure. The bases modi?ed by CMCT or DEP are represented by squares or circles, respectively. The relative extents of modi?cation are indicated by outlined stippled, outlined open, and nonoutlined shaded symbols for strong, modest, and weak reactivities, respectively. Arrowheads indicate sites cleaved by RNase V 1. C-53 was fortuitously modi?ed by DEP. Stems I, II, III, and IV are indicated, as are the initiation codon, region A, and the Shine-Dalgarno (SD) sequence. Region B (nt 1 112 to 208) is shown in boldface letters. 404 MORITA ET AL. J. B ACTERIOL. As predicted if base pairing were important, the 15A-124T double mutant carrying a compensatory mutation to restore the base pairing exhibited almost normal heat induction. The generally increased expression at both temperatures was pre- sumably due to the relative instability of A-U base pairing compared to G-C pairing.

Reexamination of the latter mutant revealed almost normal induction (data not shown); the 15C-124G mutants in the GF364 and TLF247 constructs thus gave identical results. It should be noted that a mutation within region A can af fect expression by altering complementarity to 16S rRNA (Fig. 1B) (21). The marked or slight increase found in the expression of 15A or 15C, respectively, relative to that of the wild type (Fig. 2B) was well correlated with the increased or decreased complementarity to 16S rRNA, respectively. Moreover, the higher expression of the 15A-124T double mutant than of the 15C-124G double mutant as well as the lower expression of the 17C-122G double mutant may be partially explained on the same basis. Taken together, these results con?rmed the validity of the regulatory model based on the rpoH mRNA secondary structure. However, to support this model, it was necessary to demonstrate the existence of the proposed structure. FIG. 3— Continued. V OL. 181, 1999 TRANSLATIONAL CONTROL OF s 32 SYNTHESIS 405 In general, bases strongly modi?ed by chemical probes were found in terminal loops, whereas bases in internal loops, bulges, and branching points were modi?ed less markedly. Some of the A’s and U’s in stem I predicted to form pairings were modi?ed, albeit very weakly, suggesting that the second- ary structure in this region might be relatively unstable. With respect to stems II, III, and IV, locations of modi?ed bases were in good agreement with the predicted RNA structure, with a few exceptions. These results thus provided strong evi- dence for an rpoH mRNA secondary structure with several major stem-loops (21), which has so far been supported by mutational analyses of the expression of rpoH-lacZ gene fu- sions (36) and by evolutionary conservation of the predicted RNA secondary structure among the rpoH homologs (23). It should be noted, however, that the structure shown in Fig.

This idea was initially suggested by computer pre- diction (21) and subsequently supported by mutational analy- ses (21, 36) and structural conservation among the rpoH ho- mologs (23). Such a structure seemed most likely to restrict translation by preventing ribosome entry under nonstress con- ditions. The present results of structure probing of rpoH mRNA directly supported this model (Fig. 3). Based on the structural information, possible roles of each of the major stems that constitute the whole structure were assessed by further deletion analyses. The structure probing analyses revealed that mutations within stem II af fecting translational repression (15A and 15C) af fect not only the neighboring structures of stem II but also the structures of stem III (Table 1). The simultaneous recovery of both of these ef fects of compensatory mutations (15A-124T and 15C-124G) was well correlated with the expression and regulation of fusion proteins in vivo (Fig. 2B). This ?nding was not unexpected, because some of the partially constitutive mu- tations previously isolated from GF346 (133A, 136A, and 142A) (36) were actually localized within stem III. All of these results indicated that the stabilities of stems II and III are interdependent and that changes in stem II stability, at least those involving the mutations analyzed in this study, have par- ticularly striking ef fects on thermoregulation. The results of deletion analyses indicated that most of stem II (nt 1 27 to 111) and stem III were not indispensable for thermoregulation (36) (Fig. 4). However, as discussed below, appropriate stability or instability of the mRNA secondary structure was an essential requirement for normal regulation. In this connection, the inability to respond to heat shock was previously observed when stem III was totally deleted from the GF364 fusion (36).

Although stem III was not essential, when it was absent, certain mismatches had to be introduced to the remaining segment of RNA to substitute for its function. These combined results suggested that stem IV was not essential for thermoregulation but would serve to keep the upstream Shine-Dalgarno and adjacent regions “open” for ribosome entry. We conclude that there are two major requirements for normal rpoH thermoregulation. Besides the mRNA secondary structure, previous results suggested the possible involvement of a speci?c sequence which may provide a site for protein binding in modulating the heat induction of rpoH translation. This suggestion was based mainly on the noninducible and barely inducible phenotypes of the 15C-124G and 16G-123C mutants, respectively, each con- taining two compensatory mutations (36). Although we con- ?rmed the results for the latter mutant (16G-123C; data not shown), the 15C-124G mutant actually exhibited slightly re- duced but appreciable heat induction (Fig. 2B), eliminating the major basis for suggesting the above possibility. At present, the involvement of a trans -acting factor(s) in thermoregulation appears unlikely, although it cannot be ex- cluded. The fact that some of the nucleotides expected to form a stem I structure were modi?ed by chemical probes, albeit weakly (Fig. 3B), suggested that this region was relatively un- stable, presumably permitting the limited entry of ribosomes at a low temperature. Moreover, transcription-translation cou- pling may facilitate a productive interaction between rpoH mRNA and ribosomes because of a delay in forming the stem I structure due to the distance (ca. 180 nt) between the AUG codon and the internal region presumably required for base pairings. In any event, such a dynamic mRNA secondary struc- ture should ensure the production of low but essential basal levels of s 32 at physiological temperatures under nonstress conditions.

Mutations such as 15A or 15C may decelerate the formation of an inhibitory RNA structure, thereby permitting ribosome entry and constitutively high expression even at low temperatures. Finally, s S encoded by the rpoS gene is another global reg- ulator for a set of genes induced at the stationary phase or upon hyperosmotic stress. Interestingly, s S itself is regulated primarily at the posttranscriptional level, and recent work in- dicated the involvement of some speci?c gene products in the V OL. 181, 1999 TRANSLATIONAL CONTROL OF s 32 SYNTHESIS 409 In addition, the rpoS mRNA secondary structure was suggested to play a regulatory role, although the mechanism remains unknown. Thus, trans- lational control of global transcription factors such as s 32 and s S appears to be mediated by an mRNA secondary structure and confers an ef ?cient means for a rapid response to heat or other stress. The results reported here also raise the intriguing possibility that a 5 9 portion of the rpoH mRNA secondary structure is involved in direct sensing and responding to high temperatures by enhancing ribosome entry and translation ini- tiation, leading to a rapid increase in the s 32 level and the induction of heat shock proteins. Further work is in progress to examine such possibilities. ACKNOWLEDGMENTS We are grateful to T. Linn for the kind gift of the l TLF97-3 vector and to M. Nakayama, H. Kanazawa, and M. Ueda for technical assis- tance. This work was supported in part by grants from the Japan Health Sciences Foundation, Tokyo. IRL Press, Oxford, England. 4. Craig, E. A., and C. A. Gross. 1991. Is hsp70 the cellular thermometer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 10. Gof f, S. A., and A. L. Goldberg. 1985. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. ASM Press, Washington, D.C. 14. Jaeger, J. A., D. H. Turner, and M. Zuker. 1990. Predicting optimal and suboptimal secondary structure for RNA.

American Society for Microbi- ology, Washington, D.C. 26. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 27. Sprengart, M. L., H. P. Fatscher, and E. Fuchs. 1990. The initiation of translation in E. coli: apparent base pairing between the 16S rRNA and downstream sequences of the mRNA.On the other hand, the application of several different formulations and manufacturing techniques may create a bacterial protective environment. In cheese, the persistent behavior of Shiga toxin-producing Escherichia coli (STEC) relies on complex mechanisms that enable bacteria to respond to stressful conditions found in cheese matrix. In this review, we discuss how STEC manages to survive to high and low temperatures, hyperosmotic conditions, exposure to weak organic acids, and pH decreasing related to cheese manufacturing, the cheese matrix itself and storage. Moreover, we discuss how these stress responses interact with each other by enhancing adaptation and consequently, the persistence of STEC in cheese. Further, we show how virulence genes eae and tir are affected by stress response mechanisms, increasing either cell adherence or virulence factors production, which leads to a selection of more resistant and virulent pathogens in the cheese industry, leading to a public health issue. View Show abstract. Although much attention has been paid to the role of highly conserved heat shock proteins such as chaperones and proteases in sustaining cellular protein homeostasis under stress, relatively little is known about the dynamic nature of underlying regulatory mechanisms. When cells are suddenly exposed to high temperature, synthesis of ??? is rapidly induced by activated translation of rpoH mRNA, which encodes ???, through disruption of mRNA secondary structure. The increased synthesis of ??? is accompanied by stabilization of ???

, which is normally very unstable and rapidly degraded by the membrane-localized FtsH protease. It was recently found that ??? must be localized to the inner membrane by the SRP-dependent pathway to work properly for regulation, but the roles played by membrane and other components of the cell remained unknown. Random transposon mutagenesis of the strongly deregulated I54N-??? mutant has now started to unravel the complex regulatory circuit, involving membrane protein(s), other cellular components or ???-interfering polypeptides, for dynamic fine-tuning of ??? activity that could be of vital importance for cell survival. View Show abstract.However, the full complement of these elements is not known even in the model bacterium Escherichia coli. Using complementary RNA-sequencing approaches, we detected large numbers of 3' ends in 5' UTRs and open reading frames (ORFs), suggesting extensive regulation by premature transcription termination. We document regulation for multiple transcripts, including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump. In addition to discovering novel sites of regulation, we detected short, stable RNA fragments derived from 5' UTRs and sequences internal to ORFs. Characterization of three of these transcripts, including an RNA internal to an essential cell division gene, revealed all have independent functions as sRNA sponges. Thus, these data uncover an abundance of cis- and trans-acting RNA regulators in bacterial 5' UTRs and internal to ORFs. View Show abstract. More than 200 nucleotides of the coding region of rpoH, divided into two segments, are involved in the temperature response of the transcript. Furthermore, the Shine-Dalgarno sequence in the rpoH mRNA is, unlike to the majority of described RNATs, not completely blocked (Morita et al. 1999). One of the most abundant sequences present in the 5?