Supplementary Files:
http://www.nature.com/nsmb/journal/v12/n4/suppinfo/nsmb917_S1.html

Amy E Trotochaud & Karen M Wassarman

Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, Wisconsin 53706, USA.

Correspondence should be addressed to Karen M Wassarman
E-mail:  wassarman@bact.wisc.edu

6S RNA, a highly abundant noncoding RNA, regulates transcription through interaction with RNA polymerase in Escherichia coli. Computer searches identified 6S RNAs widely among g-proteobacteria. Biochemical approaches were required to identify more divergent 6S RNAs. Two Bacillus subtilis RNAs were found to interact with the housekeeping form of RNA polymerase, thereby establishing them as 6S RNAs. A third B. subtilis RNA was discovered with distinct RNA polymerase-binding activity. Phylogenetic comparison and analysis of mutant RNAs revealed that a conserved secondary structure containing a single-stranded central bulge within a highly double-stranded molecule was essential for 6S RNA function in vivo and in vitro. Reconstitution experiments established the marked specificity of 6S RNA interactions for s⁷⁰-RNA polymerase, as well as the ability of 6S RNA to directly inhibit transcription. These data highlight the critical importance of structural characteristics for 6S RNA activity.

Figure 1. Analysis of total and immunoprecipitated RNAs.

(a) Total RNA (lanes 2-4) or immunoprecipitated RNAs (lanes 5-8) were separated on a 10% (w/v) denaturing polyacrylamide gel after 3' end labeling. Total RNA was isolated from exponential E. coli (Ec) and B. subtilis (Bs) or from plates of B. pertussis (Bp). Immunoprecipitation was done from extract of exponential B. subtilis using sera specific for the a subunit of RNAP (a) or preimmune sera (pre), monoclonal antibodies specific for s^A (s), or no antibody (-). 5' end-labeled MspI-digested pBR322 DNA (lane 1). Asterisk denotes a nonspecific band observed in immunoprecipitated samples even with no antibody.

(b) Coimmunoprecipitated RNA was analyzed by northern blot using full-length B. subtilis 6S-1 RNA-, 6S-2 RNA- or 5S RNA-specific probes. Immunoprecipitations were done with extracts of stationary- or exponential-phase B. subtilis using antibodies specific for the RNAP subunits: s^A (s, lanes 3 and 6) or a (a, lanes 2 and 5), or preimmune sera (pre, lanes 4 and 7). Total RNA (lanes 1 and 8) isolated from 30% of input extract is included for comparison. A total of 30-40% of 6S-1 RNA doublet coimmunoprecipitated with RNAP from exponential and stationary phase. About 50% of 6S-2 RNA coimmunoprecipitated with RNAP from extracts of exponential cells, and ~20% from stationary cells.

Figure 2: Selected secondary structure predictions for 6S RNAs from diverse bacteria.

Figure 2. Selected secondary structure predictions for 6S RNAs from diverse bacteria.
Predicted secondary structures were generated for each 6S RNA using mfold [7, 8] (see Methods). Structures shown were chosen to illustrate diversity in species represented as well as the 6S-1 and 6S-2 RNAs from B. subtilis.

Figure 3: Analysis of mutant 6S RNA activity by coimmunoprecipitation of s⁷⁰ with core RNAP.

Figure 3. Analysis of mutant 6S RNA activity by coimmunoprecipitation of s⁷⁰ with core RNAP.

(a) Schematic representation of mutant RNAs compared with E. coli wild type in predicted secondary structures. Altered regions are highlighted with gray boxes.

(b) RNAP was immunoprecipitated with core-specific antisera from extract of wild-type (WT) cells (lane 1), ssrS1 cells (lane 2), or ssrS1 cells with pKK* (lane 3), pKK*-6S+Y (lane 4), pKK*-6S(M5)+Y (lane 5) or pKK*-6S(M6)+Y (lane 6). Western blot analysis was done using core- (beta and beta' subunits shown) and s⁷⁰-specific antisera.

Figure 4: Examination of 6S RNA and mutants for inhibition of transcription in vivo and in vitro.

Figure 4. Examination of 6S RNA and mutants for inhibition of transcription in vivo and in vitro.

(a) 6S RNA or mutant RNAs were expressed from pKK*-6S+Y plasmid or derivatives in ssrS1 cells containing an rsdP2:lacZ reporter fusion. 6S RNA activity was measured as b-galactosidase activity and normalized to wild-type strain background (asterisk). Open-bar, wild-type strain; gray bars, ssrS1 strain; none, no vector; vector, vector control (pKK*); WT, wild type.

(b) Transcription of rsdP2 promoter from pBS-rsd1 was analyzed in extract from stationary ssrS1 cells after reconstitution with 6S RNA (lanes 1-4), 6S(M5) RNA (lanes 5-8), 5S RNA (lanes 9-12) or no RNA (lane 13). Added RNAs were at 1X (lanes 4, 8 and 12), 2X (lanes 3, 7 and 11), 4X (lanes 2, 6 and 10) or 8X (lanes 1, 5 and 9) estimated endogenous levels in wild-type cells. Transcription products were examined by RNase protection assay.

Figure 5: In vitro reconstitution to examine specificity of 6S RNA interactions with various forms of RNAP.

Figure 5. In vitro reconstitution to examine specificity of 6S RNA interactions with various forms of RNAP.

An equimolar mixture of ³²P-labeled 6S RNA, 6S(M5) RNA and 5S RNA was incubated with purified RNAP (Es⁷⁰, lanes 2-4; core, lanes 5-7; Es^S, lanes 8-10; Es³², lanes 11-13; or free s⁷⁰, lanes 14-16) in the presence of 10 mg nsRNA (lanes 3, 6, 9, 12 and 15), 75 mg ml^-1 heparin (lanes 4, 7, 10, 13 and 16) or nothing (lanes 2, 5, 8, 11 and 14), followed by immunoprecipitation using core-specific antisera (lanes 2-13) or s⁷⁰-specific antibodies (lanes 14-16). Input is the RNA mixture without incubation with RNAP, and represents 20% RNA level used for each immunoprecipitation reaction.

Figure 6: Analysis of 6S and mutants for Es⁷⁰-binding activity.

Figure 6. Analysis of 6S and mutants for Es⁷⁰-binding activity.
Gel shift analysis to measure RNA-binding activity after reconstitution with purified Es⁷⁰. RNA identities are indicated at the top of each lane. Minor bands observed with 6S(M9) RNA probably represent minor levels of alternatively folded RNAs as they were present in free RNA (data not shown).

Supplement Figure 2c: RNase mapping of secondary structure for 6S RNA and mutant RNAs.

Supplementary Figure 2: RNase mapping of secondary structure for 6S RNA and mutant RNAs.

In vitro transcribed RNAs were 3´ end labeled, gel purified, and folded prior to use.

c. Schematic representation of wild type  6S RNA in the predicted secondary structure with locations of nuclease sensitivity indicated. Circled nucleotides indicate locations of digestion by single-strand specific RNase A and RNase T1, the thickness of the lines indicates relative sensitivity. Arrows indicate locations of digestion by RNase V1. Note that data was compiled from several experiments similar to those shown in (a) and (b), in addition to RNase A and T1 analysis of 5´ end labeled 6S RNA. Therefore, not all nucleotides indicated as sensitive in (c) are readily observed in the gels shown.

Nuclease mapping experiments are consistent with our proposed 6S RNA structure with the single-stranded region and bulged nucleotides showing preferential digestion with RNase A and T1 in contrast to the absence of cleavage sites within highly double-stranded regions. Likewise, RNase V1 preferentially cleaved double-stranded regions of the RNAs. Note that RNase V1 also cleaves at stacked nucleotides which might account for the recognition of A149 and A104. A region in the bottom sequence is recognized by RNases A, T1, and V1 (C132, C133, U134, U135, G136) which also might indicate stacked nucleotides within this region.

The predicted structures of mutant 6S RNAs are strongly supported by RNase digestion. The patterns of cleavage are very similar except where sequence changes allow recognition by different RNases [such as nucleotides indicated by * for 6S(M21+M22) RNA] or when sequences are missing [such as region indicated by ** for 6S(M5) RNA]. 6S(M5) RNA shows enhanced digestion of G136, perhaps due to distortion of the single-stranded bottom region when it is opposite the minimal top region.

Additional References:

1. Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G, Sementchenko V, Piccolboni A, Bekiranov S, Bailey DK, Ganesh M, Ghosh S, Bell I, Gerhard DS, and Gingeras TR, "Transcriptional Maps of 10 Human Chromosomes at 5-Nucleotide Resolution".

2. Kiyosawa H, Mis N, Iwase S, Hayashizaki Y,  and Abe K, "Disclosing hidden transcripts: Mouse natural sense-antisense transcripts tend to be poly(A) negative and nuclear localized".

3. Storz G, Altuvia S, and Wassarman KM, "An Abundance of RNA Regulators".

4. Buskirk AR, Landrigan A, and Liu DR, "Engineering a Ligand-Dependent RNA Transcriptional Activator".

5. Hovsepian JA, and Frenster JH, "Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription".

6. Luo Y, Kurz J, MacAfee N,  and Krause MO, "C-myc Deregulation during Transformation Induction: Involvement of 7SK RNA", J. Cellular Biochemistry, vol. 64, no. 2, pp. 313-327 (1997).

7. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo", Nature, vol. 197, no. 4872, pp. 1077-1080, (March 16, 1963).

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