Regulatory studies of gene expression have traditionally concentrated on transcription initiation, but the processes of elongation, termination, and antitermination have become increasingly important aspects of gene regulation to address (2, 3, 15, 17). In particular, transcription antitermination (modification of the transcription apparatus so that terminators can be bypassed) presents an interesting regulatory mechanism. Transcription and translation are coupled for most operons of Escherichia coli. However, when translation is arrested or untranslated regions of RNA are synthesized early termination of transcription often results, causing a dramatic decrease in downstream gene expression, a phenomenon known as polarity (17). Untranslated regions of RNA frequently permit access by the transcription termination protein, Rho, leading to premature termination of transcription and release of RNA polymerase from the transcript (for a review, see reference 17). The transcripts of rRNA operons are not translated and contain Rho-dependent termination sites but are not subject to polarity (1, 5, 14). The absence of polarity and the synthesis of stoichiometric amounts of all three rRNA subunits are accounted for by a specific type of transcription antitermination (4, 10). Rho-dependent terminator suppression in the rRNA operons is mediated by special antiterminator sequences that occur once in the leader region and again in the spacer region between the 16S and 23S genes (4, 8, 10, 18). These sequences can effectively suppress a variety of Rho-dependent terminators both in vivo and in vitro (10, 20). An important part of the rRNA antiterminator is the sequence GCTCTTTAACAA, called boxA (4). Host proteins NusA, NusB, NusE (ribosomal protein S10), and NusG are thought to be involved, and additional cellular proteins such as ribosomal protein S4 are also required for efficient terminator readthrough (20, 22). Several of these host factors interact directly with RNA polymerase, rendering it resistant to Rho-dependent termination (11-13, 22). Exactly how they accomplish this task is not known. A further unanswered question is whether the nature of the σ subunit associated with RNA polymerase affects antitermination. For example, if a particular σ factor did not cycle off after initiation, RNA polymerase might not be able to recruit other factors necessary for the alteration of its transcription properties. When cells are subjected to stresses such as rapid heat or osmotic changes, selective groups of proteins are rapidly and transiently induced to protect the cell or help it adapt to the new environment. The heat shock response results when RNA polymerase associates with an alternative σ factor, σ32, which directs core RNA polymerase to distinct promoters (7, 23). The consensus sequences of heat shock gene promoters differ considerably from those of σ70-dependent promoters. There is no evidence that these promoters undergo cross-recognition (a mechanism which provides heat shock genes with regulation distinct from that of most of cellular proteins) (7, 25) either in vivo and in vitro. In the present study, we tested whether the antitermination properties of RNA polymerase are altered as a result of initiation at heat shock promoters. It is particularly interesting that all rRNA operon P1 promoters have interdigitated heat shock promoters. Both the σ70-dependent and σ32-dependent promoters initiated RNA transcription at the same nucleotide (16). To measure the terminator readthrough properties of RNA polymerase molecules initiated at these promoters, we used gene fusion plasmids to construct an antitermination assay system (Fig. (Fig.1A).1A). Each promoter sequence and its position relative to the boxA feature of the antiterminator sequence are shown in Fig. Fig.1B,1B, and their relevant structures with respect to promoters, antiterminators, and terminators are listed in Table Table1.1. Plasmid pSL100 was used as the parental plasmid for all constructs. Its structure and those of pSL102 and pSL103 (containing rrnGP2 [the σ70 promoter]) have been detailed by Li et al. (10). A fragment containing the groEP heat shock promoter was obtained from plasmid pDC440 (7) by digestion with TaqI and HpaI. The isolated fragment was ligated into pSL102 and pSL103 digested with ClaI to yield pSGE102 and pSGE103, respectively. The heat shock promoter from the clpB gene was amplified from plasmid pClpB (21) by PCR with a 5′ BglII site and a 3′ ClaI site and used to replace the rrnGP2 fragment of pSL102 and pSL103, resulting in pSCB102 and pSCB103, respectively. The heat shock promoter from the dnaK gene was cloned from pDC403 (7). FIG. 1. Gene fusion plasmid antitermination assay system and sequences of test promoters. (A) A reporter gene, CM acetyltransferase (cat), was placed down stream of a Rho-dependent terminator, Ter. Open boxes represent the cat gene and the bla gene (encoding ... TABLE 1. Plasmids used in this study A promoter containing HpaII and HinPI fragments was inserted into the ClaI sites of pSL100 and pSL101 (10) to yield pSDK102 and pSDK103, respectively. rrn antitermination sequences were obtained (using 5′ and 3′ BamHI primers) from the pRATT1 plasmid (20). The amplified fragment was digested with BamHI and purified. This antiterminator-containing fragment was then ligated into previously constructed plasmids (except pSDK102 and pSDK103) digested with BamHI. The antiterminator sequence was inserted in either the correct or an inverse orientation. The source of antitermination rrnG leader sequences for pSDK114 and pSDK115 was pSL104 (10), which was cleaved with BglII and TaqI and inserted into pSL102 and pSL103 (10). The Rho-dependent terminator sequence (16S←) used was a HindIII 16S fragment from the rrnB operon inserted into pSL100 in the backwards orientation to yield pSL103. In this orientation, the fragment fortuitously contains a strong Rho-dependent terminator (10). The sequences of promoters and the orientation of the antiterminator sequence were verified by DNA sequencing using a Perkin-Elmer Prism sequencer (Perkin-Elmer, Boston, Mass). E. coli strain MC1009 [Δ(lacIPOZY) galU galK Δ(ara-leu) rpsL srl::Tn10 recA spoT relA] (20) was the host strain for all plasmids used in this study. Strains harboring test plasmids were used to inoculate 6 ml of Luria broth supplemented with 1% glucose and 100 μg of ampicillin/ml from overnight cultures in the same medium and incubated with shaking at 37°C. When the culture density reached an optical density at 600 nm of 1.0, cells were harvested by centrifugation in a microcentrifuge. The cell pellets were then frozen in a dry ice-ethanol bath and kept at −80°C. RNA isolation was done using an RNeasy RNA isolation kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The concentration of total RNA was measured using absorbance at 260 nm and kept at −80°C until further analysis was performed. Two end-labeled oligonucleotide probes were used to quantitate mRNA levels: (i) a chloramphenicol (CM) acetyltransferase (cat) probe (5′-TGCCATTGGGATATATCAACGGTGG-3′) (located at nucleotides 26 to 50 of the cat gene encoding sequence and used to measure cat gene expression) and (ii) β-lactamase (bla) probe (5′-GGGAATAAGGCGACACGGAAATG-3′) (located at nucleotides 13 to 36 of the bla gene encoding sequence and used to quantitate the level of bla gene expression). This measurement serves as an internal control to correct for variations in sample preparation and plasmid copy number (9). Slot blot analyses were carried out in triplicate for each sample of 5 μg of denatured total RNA by the method described by Zellars and Squires (24).