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 are known to rely on RNA structure and RNA binding proteins in controlling 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 control mechanisms. When activated by tryptophan, TRAP binds to a segment of the trp leader transcript and prevents formation of an RNA secondary structure, the antiterminator, thereby allowing formation of an overlapping transcription terminator that causes transcription to terminate 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 factor NusA participates 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 sequesters the trpE ribosome binding site such that ribosomes are unable to initiate translation. We recently found that 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 each case, TRAP directly binds to the cognate ribosome binding site, thereby preventing translation initiation. Thus, TRAP coordinately regulates tryptophan biosynthesis, tryptophan transport and efflux of an unknown compound(s) by five related but distinct mechanisms.

TRAP is a complex consisting of 11 identical subunits. Association of tryptophan to TRAP involves extensive hydrogen bond formation. The TRAP binding site in the trp leader consists of 11 (G/U)AG repeats while the trpG and trpP and ycbK binding sites each contain 9 repeats. Each tryptophan-activated TRAP subunit can bind to one trinucleotide repeat in a target transcript, thereby wrapping the RNA around the periphery of the TRAP complex.

Bacteria have evolved several regulatory strategies that ensure their survival during the transition from exponential growth into stationary phase. The global carbon storage regulatory system (Csr) is involved in the repression of several stationary phase processes and in the activation of some exponential phase functions. Four major components of Csr include an RNA binding protein (CsrA), two small regulatory RNA molecules (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 function as antagonists of CsrA by sequestering this protein. Expression of the two regulatory RNAs is under control of the BarA-UvrY two-component signal transduction system.

CsrA regulates glycogen biosynthesis by binding to multiple sites in the glgC transcript, thereby inhibiting ribosome binding. Similar translation repression mechanisms regulate genes involved in peptide transport, quorum sensing and biofilm formation. In contrast, CsrA is a positive regulator of flagella biosynthesis, although this regulatory mechanism remains to be determined. We are using bioinformatics and genomic approaches to identify other genes and processes under control of the Csr system.

We are also investigating the role of CsrA in B. subtilis. Expression of B. subtilis csrA is increased several fold during the transition from exponential to stationary phase growth. CsrA represses hag translation, the gene encoding the B. subtilis flagellin protein, by a mechanism similar to the repression mechanisms identified in E. coli.

Representative Publications

• Weilbacher,Thomas, Kazushi Suzuki, Ashok K. Dubey, Xin Wang, Seshigirao Gudapaty, Igor Morozov, Carol S. Baker, Dimitris Georgellis, Paul Babitzke, and Tony Romeo. (2003) A novel sRNA component of the carbon storage regulatory system of Escherichia coli. Mol. Microbiol. 48:657-670.
• Dubey, Ashok K., Carol S. Baker, Kazushi Suzuki, A. Daniel Jones, Pallavi Pandit, Tony Romeo, and Paul Babitzke. (2003) CsrA regulates translation of the Escherichia coli carbon starvation gene, cstA, by blocking ribosome access to the cstA transcript. J. Bacteriol. 185:4450-4460.
• Schaak, Janell, Helen Yakhnin, Philip C. Bevilacqua, and Paul Babitzke. (2003) A Mg²⁺-dependent RNA tertiary structure forms in the Bacillus subtilis trp operon leader transcript and appears to interfere with trpE translation control by inhibiting TRAP binding. J. Mol. Biol. 332:555-574.
• Schaak, Janell, Paul Babitzke, and Philip C. Bevilacqua. (2003) Phylogentic consevation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus.  RNA 9:1502-1515.
• Babitzke, Paul, Janell Schaak, Alexander V. Yakhnin, and Philip C. Bevilacqua. (2003) Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System. Methods Enzymol. 371:392-404.
• Yakhnin, Helen, Hong Zhang, Alexander V. Yakhnin, and Paul Babitzke. (2004) The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis regulates translation of the tryptophan transport gene, trpP (yhaG), by blocking ribosome binding. J. Bacteriol. 186:278-286.
• Deikus, Gintaras, Paul Babitzke, and David H. Bechhofer. (2004) Recycling of an RNA-binding protein by ribonuclease digestion. Proc. Natl. Acad. Sci. USA 101:2747-2751.
• Babitzke, Paul. (2004) Regulation of transcription attenuation and translation initiation by allosteric control of an RNA binding protein: the Bacillus subtilis TRAP protein. Curr. Opin. Microbiol. 7:132-139.
• Yakhnin, Helen, and Paul Babitzke. (2004) Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. Appl. Microbiol. Biotechnol. 64:382-386.

• Wang, Xin, Ashok K. Dubey, Kazushi Suzuki, Carol S. Baker, Paul Babitzke, and Tony Romeo. (2005) CsrA post-transcriptionally represses pgaABCD, responsible for the synthesis of a biofilm polysaccharide adhesin of Escherichia coli. Mol. Microbiol. 56:1648-1663.

• Gollnick, Paul, Paul Babitzke, Alfred Antson, and Charles Yanofsky (2005) Complexity in regulation of tryptophan biosynthesis in Bacillus subtilis. Ann. Rev. Genet. 39:47-68.

• Dubey, Ashok K., Carol S. Baker, Tony Romeo, and Paul Babitzke. (2005) RNA sequence and secondary structure participate in High-affinity CsrA-RNA interaction. RNA 11:1579-1587.

• 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).

Search for the MEDLINE database at PubMed for articles by P Babitzke