Paul Babitzke Professor of Biochemistry and Molecular Biology 308 S. Frear Laboratory, University Park, PA 16802 Phone: (814) 865-0002 Fax: (814) 863-7024 E-mail: pxb28@psu.edu B.S. in biomedical science, St. Cloud State University Ph.D. in genetics, University of Georgia Postdoctoral, Stanford University Babitzke Lab Web Site

Regulation of Prokaryotic Gene Expression by RNA Structure and RNA-binding Proteins

Several prokaryotic and eukaryotic genes rely on RNA structure and RNA binding proteins in regulating their expression, indicating that the crucial regulatory events take place after transcription initiation. The long-term goal of my laboratory is to identify regulatory systems that involve RNA structure and RNA binding proteins, and to elucidate the underlying regulatory mechanisms.

The Bacillus subtilis trpEDCFBA operon is regulated by TRAP-mediated transcription attenuation and translation repression mechanisms. When activated by tryptophan, TRAP binds to to the nascent trp leader transcript and prevents formation of an RNA secondary structure, the antiterminator, thereby allowing formation of an overlapping transcription terminator.  Thus, transcription halts before RNA polymerase reaches the trp structural genes. In the absence of tryptophan TRAP does not bind to the message. In this case, formation of the antiterminator allows transcription readthrough into the structural genes. In addition to TRAP, the general transcription elongation factors NusA and NusG participate in the attenuation mechanism. NusA stimulates RNA polymerase pausing at the nucleotide just preceding the critical overlap between the antiterminator and terminator structures. Thus, NusA-stimulated pausing provides additional time for TRAP to bind to the nascent trp transcript and promote termination.

TRAP also regulates translation of the trp operon. TRAP binding to trp operon readthrough transcripts promotes formation of an RNA secondary structure that prevents ribosome binding. RNA polymerase pausing plays a vital role in the translation control mechanism. Interestingly, both NusA and NusG stimulate pausing at a position just downstream from the trp leader terminator. In this case, RNA polymerase pausing provides additional time for TRAP binding and to ensure that the RNA folds into the proper structure. The unlinked tryptophan biosynthetic gene (trpG), a tryptophan transport gene (trpP), and an efflux gene of unknown function (ycbK) are also regulated by TRAP. In these three cases, TRAP directly binds to the cognate ribosome binding site, thereby preventing translation initiation. Thus, TRAP coordinately regulates tryptophan metabolism by three distinct mechanisms.

Cognate intrinsic transcription terminators consist of a contiguous RNA hairpin followed by a U-rich tract. Our recent work indicates that NusA is required for termination at non-cognate terminators that have interrupted hairpins and/or those with a minimal U-rich tract. Thus, NusA-dependent termination constitutes a previously unrecognized transcription termination mechanism in bacteria. Our current work in B. subtilis continues to focus on the mechanism of RNA polymerase pausing and transcription termination.

A second major effort in the lab involves genetic and biochemical characterization of the global regulatory system called Csr. Bacteria have evolved several regulatory strategies that ensure their survival during the transition from exponential growth into stationary phase. The Csr system represses or activates approximately 700 E. coli genes, thereby mediating global changes in cellular physiology during the transition between exponential and stationary phase growth. Four major components of Csr include an RNA binding protein (CsrA), two small RNA (sRNA) antagonists of CsrA (CsrB and CsrC), and CsrD, a protein that targets degradation of CsrB and CsrC by RNase E. CsrA represses gluconeogenesis, glycogen biosynthesis and catabolism, biofilm formation and quorum sensing. Conversely, CsrA activates glycolysis, acetate metabolism and flagella biosynthesis. CsrB and CsrC RNAs contain multiple CsrA binding sites, and are capable of sequestering several molecules of CsrA.

CsrA represses translation of numerous genes by binding to the translation initiation regions of these genes. Bound CsrA prevents ribosome binding, thereby repressing a variety of processes, including glycogen biosynthesis, peptide transport, quorum sensing and biofilm formation. In contrast, CsrA is a positive regulator of flagella biosynthesis. In this case bound CsrA stabilizes the mRNA by preventing degradation by RNase E. We continue to explore this global regulatory system from a systems biology point of view.

Representative Publications

• Yakhnin, Helen, Alexander V. Yakhnin, and Paul Babitzke. (2006) The trp RNA-binding Attenuation Protein (TRAP) of Bacillus subtilis Regulates Translation Initiation of ycbK, a Gene Encoding a Putative Efflux Protein, by Blocking Ribosome Binding. Mol. Microbiol. 61:1252-1266.

• Suzuki, Kazushi, Paul Babitzke, Sidney R. Kushner, and Tony Romeo. (2006) Identification of a Novel Regulatory Protein (CsrD) that Targets the Global Regulatory RNAs CsrB and CsrC for Degradation by RNase E. Genes Dev. 20:2605-2617.

• Mercante, Jeffrey, Kazushi Suzuki, Xiaodong Cheng, Paul Babitzke, and Tony Romeo (2006) Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance. J. Biol. Chem. 281:31832-31842.

• Yakhnin, Alexander V., Helen Yakhnin, and Paul Babitzke (2006) RNA Polymerase Pausing Participates in the Bacillus subtilis trpE Translation Control Mechanism by Providing Additional Time for TRAP to Bind to the Nascent trp Leader Transcript. Mol. Cell 24:547-557.

• Yakhnin, Helen, Alexander V. Yakhnin, and Paul Babitzke. (2007) Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not. J. Bacteriol. 189:872-879.

• Babitzke, Paul, and Tony Romeo (2007) CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr. Opin. Microbiol. 10:156-163.

• Yakhnin, Helen, Pallavi Pandit, Tom J. Petty, Carol S. Baker, Tony Romeo, and Paul Babitzke. (2007) CsrA of Bacillus subtilis regulates translation initiation of the gene encoding the flagellin protein (hag) by blocking ribosome binding. Mol. Microbiol. 64:1605-1620.
• Baker, Carol S., Lél A. Eöry, Helen Yakhnin, Jeffrey Mercante, Tony Romeo, and Paul Babitzke (2007). CsrA inhibits translation initiation of Escherichia coli hfq by binding to a single site overlapping the Shine-Dalgarno sequence. J. Bacteriol. 189:5472-5481.

• Lapouge, Karine, Elena Sineva, Magnus Lindell, Katja Starke, Carol S. Baker, Paul Babitzke, and Dieter Haas. (2007) Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens. Mol. Microbiol. 66:341-356.

• McGraw, Adam, Philip C. Bevilacqua, and Paul Babitzke. (2007) TRAP-5' Stem-Loop Interaction Increases the Efficiency of Transcription Termination in the Bacillus subtilis trpEDCFBA Operon Leader Region. RNA (In Press).

• Babitzke, Paul, and Paul Gollnick. (2008) Control of gene expression by attenuation. In The Wiley Encyclopedia of Chemical Biology, T Begley, ed. John Wiley & Sons, Inc., Hoboken, NJ, Vol. 1, pp. 147-158.

• Yakhnin, Alexander V., Helen Yakhnin and Paul Babitzke (2008) Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader. Proc. Natl. Acad. Sci. USA 105:16131-16136.

• McGraw, Adam P., Ali Mokdad, François Major, Philip C. Bevilacqua, and Paul Babitzke. (2009) Molecular basis of TRAP-5’SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism. RNA 15:55-66.
• Babitzke, Paul, Carol S. Baker, and Tony Romeo. (2009) Regulation of translation initiation by RNA binding proteins. Ann. Rev. Microbiol. 63:27-44.
• Mercante, Jeffrey, Adrianne N. Edwards, Ashok K. Dubey, Paul Babitzke, and Tony Romeo. (2009) Molecular geometry of CsrA (RsmA) binding to RNA and its implications for regulated expression. J. Mol. Biol. 392:511-528.
• Romeo, Tony, and Paul Babitzke. (2009) Csr (Rsm) system and its overlap and interplay with c-di-GMP regulatory systems. In The Second Messenger Cyclic Di-Guanylate, ed. A. Wolfe and K. Visick. Washington, D.C.:American Society for Microbiology (In Press).
• Perez, Nataly, Jeanette Treviño, Zhuyun Liu, Siu Ho, Paul Babitzke, and Paul Sumby. (2009) A genome-wide analysis of small regulatory RNAs in the human pathogen group A Streptococcus. PLoS ONE (In Press).
• Yakhnin, Alexander V., and Paul Babitzke (2009) Mechanism of NusG-stimulated pausing, hairpin-dependent pause site selection and intrinsic termination at overlapping pause and termination sites in the Bacillus subtilis trp leader. (Submitted).

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