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Rho Directs Widespread Termination of Intragenic and Stable RNA Transcription Jason M

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Rho Directs Widespread Termination of Intragenic and Stable RNA Transcription Jason M Rho directs widespread termination of intragenic and stable RNA transcription Jason M. Petersa,b, Rachel A. Mooneya, Pei Fen Kuanc, Jennifer L. Rowlanda,Su¨ ndu¨z Keles¸c,d, and Robert Landicka,e,1 Departments of aBiochemistry, bGenetics, cStatistics, dBiostatistics and Medical Informatics, and eBacteriology, University of Wisconsin, Madison, WI 53706 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved July 16, 2009 (received for review April 8, 2009) The transcription termination factor Rho is a global regulator of RNA levels. Their findings showed changes in abundance of a subset of polymerase (RNAP). Although individual Rho-dependent terminators transcripts, particularly for genes integrated into the genome by have been studied extensively, less is known about the sites of RNAP horizontal transfer. Thus, Rho termination occurs preferentially on regulation by Rho on a genome-wide scale. Using chromatin immu- a subset of genes, even though its physical distribution is wide- noprecipitation and microarrays (ChIP-chip), we examined changes in spread. However, the specific locations of BCM-inhibited Rho- the distribution of Escherichia coli RNAP in response to the Rho- dependent terminators have not yet been determined. specific inhibitor bicyclomycin (BCM). We found Ϸ200 Rho-terminated We used ChIP-chip to examine changes in the distribution of loci that were divided evenly into 2 classes: intergenic (at the ends of RNAP in response to Rho inhibition by BCM. We found Ϸ200 genes) and intragenic (within genes). The intergenic class contained Rho-terminated loci where BCM shifted the distribution of noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs RNAP downstream of the apparent termination site. Half of the (tRNAs), establishing a previously unappreciated role of Rho in ter- Rho-dependent terminators were located at the 3Ј ends of genes mination of stable RNA synthesis. The intragenic class of terminators (intergenic), including small RNAs (sRNAs) and transfer RNAs included a previously uncharacterized set of short antisense tran- (tRNAs). The other half were found within the coding sequence scripts, as judged by a shift in the distribution of RNAP in BCM-treated of annotated genes (intragenic). For one set of intragenic cells that was opposite to the direction of the corresponding gene. terminators, the readthrough event was in the opposite direction These Rho-terminated antisense transcripts point to a role of non- of the gene, indicating antisense transcription. coding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad Results roles in eukaryotic gene regulation. BCM Alters the Distribution of RNAP. To determine the contribution of Rho to the genome-wide distribution of RNAP, ChIP was performed chromatin immunoprecipitation ͉ RNA polymerase on cells grown in the presence or absence of BCM at 20 ␮g/mL. This concentration of BCM was chosen because it did not alter the growth ranscription termination is critical for maintaining control rate of cells under the conditions used in these experiments (14), and Tover gene expression. Bacteria employ 2 distinct types of thus limited the potential indirect effects that could result from inhib- termination: (i) intrinsic termination, for which a GC-rich RNA iting Rho. DNAs from ChIP experiments targeting the ␤ or ␤Ј subunit hairpin followed by a U-tract dissociates RNA polymerase of RNAP and ‘‘input’’ genomic DNA were differentially labeled with (RNAP) without the need for accessory proteins, and (ii) Cy3 and Cy5 dyes, then hybridized to a tiling microarray (see Materials factor-dependent termination caused by the Rho protein. Rho and Methods), revealing the RNAP distribution in BCM-treated and was originally identified as a factor that increased the ‘‘accuracy’’ untreated conditions. Independent biological replicates showed good of in vitro transcription by terminating RNAP at specific posi- agreement (Pearson’s r ϭ 0.9). tions on a bacteriophage ␭ DNA template (1). Later, Rho was Changes in the distribution of RNAP upon BCM treatment were found to be the cause of polarity, whereby the uncoupling of readily apparent by visual inspection of the data, and were quantified transcription and translation by premature stop codons de- statistically. A moving average method implemented in the program creases gene expression of downstream genes in an operon (2). CMARRT (15) was used to identify regions where at least 3 consec- Rho is a homohexameric protein with RNA-dependent ATPase utive probes exhibited increased ChIP-chip signal in BCM-treated cells activity (3). Rho binds to the nascent RNA and translocates 5Ј versus untreated cells (see Materials and Methods; no BCM-induced to 3Ј along RNA using energy derived from ATP hydrolysis (4). reductions in RNAP occupancy were detected). This analysis revealed At certain sites, Rho contacts RNAP, and terminates the a total of 199 BCM significant regions (BSRs) dispersed throughout the elongation complex (EC) by an unknown mechanism (5). E. coli K-12 chromosome. Most of the probes with increased ChIP-chip Bicyclomycin (BCM) is a specific inhibitor of Rho (6). BCM signal in BCM-treated cells were within BSRs, but they represented blocks Rho-dependent termination in vivo (7) and in vitro (8) only a small percentage of the total probes (Ϸ3%; Fig. 1A). This through noncompetitive inhibition of the RNA-dependent AT- suggests that the effects of BCM were mostly direct consequences of Pase activity of Rho (9). Biochemical and structural analyses Rho inhibition rather than a large-scale redistribution of RNAP in show that BCM binds adjacent to the ATPase of Rho (8) and response to cellular stresses or other pleiotropic effects. prevents ATP hydrolysis by interfering with a key glutamic acid The BSR dataset was compared to previously characterized Rho- residue that is involved in catalysis (10). Treatment of wild-type dependent terminators, which confirmed that BCM effectively inhib- E. coli K-12 with high concentrations of BCM is lethal (6), ited Rho in our experiment. For instance, the rho gene is autoregulated because rho is an essential gene (11). However, sublethal doses of BCM are sufficient to perturb Rho termination in vivo (7). Genome-wide studies have documented the role of Rho as a Author contributions: J.M.P., R.A.M., and R.L. designed research; J.M.P., R.A.M., and J.L.R. per- global regulator of RNAP. Chromatin immunoprecipitation assays formed research; J.M.P., P.F.K., and S.K. analyzed data; and J.M.P., S.K., and R.L. wrote the paper. using tiling microarrays (ChIP-chip) revealed remarkably similar The authors declare no conflict of interest. global distributions of RNAP and Rho on DNA (12). These similar This article is a PNAS Direct Submission. distributions suggest that Rho contacts ECs soon after initiation, Data deposition: The data reported in this paper have been deposited in the Gene interacts with ECs throughout elongation, and interacts with ECs Expression Omnibus (GEO) database, www.ncbi.nim.nih.gov/geo (accession no. GSE16562). on nearly all transcription units (TUs), rather than having speci- 1To whom correspondence should be addressed. E-mail: [email protected]. ficity for a small set of genes. Cardinale et al. (13) used expression This article contains supporting information online at www.pnas.org/cgi/content/full/ array analysis to gauge the effect of BCM treatment on mRNA 0903846106/DCSupplemental. 15406–15411 ͉ PNAS ͉ September 8, 2009 ͉ vol. 106 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903846106 Downloaded by guest on September 30, 2021 Table 1. BSR Annotation summary A 3 BSR type tRNAa sRNAb mRNAc Total K-12 specificd Prophaged 2 Intergenic 12 7 83 102 21 15 Intragenic 1 Sense 0 0 57 57 10 4 Antisense 0 0 25 25 6 3 0 NDe 0 0 15 15 2 1 Total 12 7 180 199 39 23 -1 RNAP + BCM [log2(IP / input)] aNumber of BSRs associated with annotated tRNA genes. -2-10123 bNumber of BSRs associated with annotated sRNA genes. RNAP no BCM [log2(IP / input)] cNumber of BSRs associated with annotated mRNA genes. dNumber of BSRs associated with E. coli K-12-specific genes or prophageDNA B 3 -BCM (ASAP Database, http://www.genome.wisc.edu/tools/asap.htm). +BCM eDirectionality was not determined. 2 1 array-based ChIP-chip data likely explains this discordance, al- though differences in experimental growth conditions could also RNAP [log2(IP / input)] contribute. Importantly, the ChIP-chip-derived BSR data define rhoL the locations at which Rho-dependent termination normally occurs. rho 3964000 3965000 3966000 To understand the roles of these Rho-dependent terminators, we Genome Position next sought to associate each BSR with a specific gene. Although ChIP-chip experiments do not provide strand information per se, After Genes Within Genes C (Intergenic) (Intragenic) the ‘‘directionality’’ of terminator readthough was used to assess the orientation of RNAP on DNA. An example of directionality can be 1122 22511555 found at the rho locus (Fig. 1B). The distribution of RNAP shifts 8833 7 to the right downstream of rhoL in ChIP-chip data from BCM- GENETICS 2255 treated cells compared with untreated cells. Therefore, the termi- nator must be on the ‘‘plus’’ strand at the 3Ј end of rhoL. This logic 7722 5577 was extended to assign each BSR to a particular gene (Table S1). When directionality could not be determined (as was the case in 15 Gene Type Orientation BSRs), the BSR was assigned to the gene that contained the mRNA { sense { majority of significant probes for that BSR. Quantitative PCR of K-12-specific K-12-specific ChIP DNA was used to confirm the array results at 3 of the anti- tRNA BSR-associated loci (rho, valVW, and rygD; Fig. S2). { K-12-specific sense { K-12-specific sRNA Our analysis revealed a diverse set of Rho targets in the E.
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