Jeffrey E. Barrick ¹, Narasimhan Sudarsan ², Zasha Weinberg ³, Walter L. Ruzzo ^{3, 4}, and Ronald R. Breaker ²

¹ Department of Molecular Biophysics and Biochemistry and ² Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA
³ Department of Computer Science and Engineering and ⁴ Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA

Reprint requests to: Ronald R. Breaker, Department of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520, USA;
e-mail:   ronald.breaker@yale.edu       fax: (203) 432-6604.

6S RNA is an abundant noncoding RNA in Escherichia coli that binds to s⁷⁰ RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation.

6S RNA consensus secondary structure compared with open promoter DNA.

FIGURE 2. 6S RNA consensus secondary structure compared with open promoter DNA.

(A) Consensus secondary structure model for 6S RNA. Nucleotide symbols and shading are the same as in Figure 1 (consensus line). Certain nucleotides whose identity is not conserved but are present in >60% of sequences are represented as empty circles. Solid lines represent variable regions of the structure. Three parallel insets show lineage-specific terminal loop structures and sequence conservation, and the boxed nucleotides on the 3' side of the central bubble can alternately form the pictured base-paired stem in many sequences. Annotated nucleotide distances are the median lengths between conserved segments.

(B) Schematic of DNA template in the open promoter complex with RNA polymerase (RPo) as described elsewhere (Murakami et al. 2002).

In-line probing of 6S RNA structures.

FIGURE 3. In-line probing of 6S RNA structures.

(A) Sequence, secondary structure, and in-line probing data for E. coli 6S-184 RNA. Levels of spontaneous RNA cleavage at backbone linkages within the construct depicted to the right were measured by separating 5'-radiolabeled degradation products on a polyacrylamide gel. In-line probing gel lanes are as follows: NR, no incubation; T1, partial digestion with RNase T1 (cleaves 3' of G nucleotides); –OH, partial alkaline digestion; P, spontaneous cleavage during a 40-h in-line probing reaction incubated at 25°C. Pre identifies the full-length RNA. Bands corresponding to certain T1 cleavage products are identified as position markers. In the secondary structure model, shaded circles identify nucleotides whose 3' linkage undergoes a high level of spontaneous cleavage relative to most other linkages. Filled triangles mark the extent of the region where cleavages were mapped. Lowercase letters identify unnatural guanosine nucleotides added for efficient in vitro transcription with T7 RNAP.

(B) Sequence, secondary structure, and in-line probing data for B. subtilis 6Sb-201 RNA. Details as in A. Slow in vivo processing of 201 nt 6Sb RNA cleaves off 11 nucleotides (gray), resulting in the 190-nt form of 6Sb RNA.

Additional References:

1. Trotochaud AE, and  Wassarman KM, "A highly conserved 6S RNA structure is required for regulation of transcription", Nature Structural & Molecular Biology, vol. 12, no 4, pp. 313-319 (April, 2005).

2. 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".

3. 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".

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

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

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

7. 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).

8. 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).

Links to RNA and Biological Causality:

A Brief History of Activator RNA:

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A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).