Paul Babitzke
Professor of Biochemistry and Molecular Biology
Co-director, Center for RNA Molecular Biology
University Park, PA 16802
Research Interests
Regulation of gene expression by RNA structure and RNA-binding proteins
Research Summary
Gene expression can be regulated at the level of transcription, translation and mRNA stability. The transcription cycle consists of initiation, elongation and termination, each of which can be regulated. We are investigating fundamental mechanisms that affect transcription elongation and termination. We are also investigating a variety of genes in which RNA binding proteins control gene expression by transcription attenuation (regulated termination), repression of translation initiation and/or mRNA stability.
The Bacillus subtilis trp operon is regulated by TRAP-mediated transcription attenuation and translation repression mechanisms. When activated by tryptophan, TRAP binds to the nascent trp transcript and prevents formation of an antiterminator structure, thereby allowing formation of an overlapping transcription terminator, which halts transcription before RNA polymerase reaches the trp structural genes. In the absence of tryptophan TRAP does not bind to the RNA such that formation of the antiterminator allows transcription into the structural genes. In addition to TRAP, the transcription elongation factors NusA and NusG participate in the attenuation mechanism. NusA and NusG stimulate RNA polymerase pausing at the nucleotide just preceding the critical overlap between the antiterminator and terminator structures. Thus, NusA- and NusG-stimulated pausing provides additional time for TRAP to bind to the transcript and promote termination. TRAP also regulates translation of the trp operon. TRAP binding to trp operon readthrough transcripts promotes formation of an RNA structure that prevents ribosome binding. NusA and NusG stimulated pausing plays a vital role in this mechanism. In this case, pausing provides additional time for TRAP to bind and promote formation of the RNA structure. In addition to exploring these pausing mechanisms, we are using genomic approaches to identify additional NusG-stimulated pause sites.
Canonical intrinsic transcription terminators consist of a contiguous RNA hairpin followed by a U-rich tract. We recently found that NusA is required for termination at non-canonical 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. We are continuing to investigate this phenomenon in B. subtilis and E. coli.
Another major effort in the lab involves genetic and biochemical characterization of the Csr global regulatory system. Csr regulates ~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 translation initiation of numerous genes by binding to their translation initiation regions. Bound CsrA prevents ribosome binding, thereby repressing a variety of processes, including gluconeogenesis, glycogen biosynthesis, quorum sensing and biofilm formation. In contrast, CsrA activates glycolysis, acetate metabolism and flagella biosynthesis. CsrA activates flagella biosynthesis by preventing degradation of the flhDC transcript by RNase E. We continue to explore this global regulatory system in E. coli and B. subtilis from biochemical and genomic viewpoints.
Model of the termination/pausing decision in the B. subtilis trp leader
(a) RNAP with bound NusA and NusG arrives at the termination site. In the presence of bound TRAP the antiterminator does not form. Thus, NusA-stimulated folding of the terminator/pause hairpin leads to termination (not shown). In the absence of bound TRAP the antiterminator forms, thereby preventing completion of the terminator/pause hairpin. Thus, RNAP transcribes past the terminator.
(b) RNAP with bound NusA and NusG arrives at the U144 pause site and RNAP enters into an elemental pause state. NusA-stimulated completion of the terminator/pause hairpin via branch migration stabilizes the pause. Sequence-specific interaction of NusG with T137-T139 of the non-template DNA strand further stabilizes the pause at U144.
Model of CsrA-RNA interaction
(a) Each CsrA dimer contains two symmetric RNA binding surfaces. CsrA initially interacts with a high-affinity site within the loop of a short RNA hairpin.
(b) The initial interaction increases the localized concentration of CsrA such that it can bind to a low-affinity site overlapping the target mRNA's Shine-Dalgarno (SD) sequence.
(c) Bound CsrA blocks ribosome binding, thereby repressing translation initiation.
Representative Publications
- 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.
- 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.
- 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.
- 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.
- Babitzke, Paul, Carol S. Baker, and Tony Romeo. (2009) Regulation of translation initiation by RNA binding proteins. Ann. Rev. Microbiol. 63:27-44.
- Yakhnin, Alexander V., and Paul Babitzke (2010) 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. Mol. Microbiol. 76:690-705.
- Edwards, Adrianne N., Laura Patterson-Fortin, Christopher a. Vakulskas, Jeffrey W. Mercante, Katarzyna Potrykus, Daniel Vinella, Martha I. Camacho, Joshua A. Fields, Stuart A. Thompson, Dimitris Georgellis, Michael Cashel, Paul Babitzke, and Tony Romeo. (2011) Circuitry Linking the Csr and Stringent Response Global Regulatory Systems. Mol Microbiol. 80:1561-1580.
- Martinez, Luary C., Helen Yakhnin, Martha I. Camancho, Dimitris Georgellis, Paul Babitzke, José L. Puente, and Victor H. Bustamante. (2011) Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons via HilD. Mol Microbiol. 80:1637-1656.
- Yakhnin, Helen, Alexander V. Yakhnin, Carol S. Baker, Elena Sineva, Igor Berezin, Tony Romeo, and Paul Babitzke. (2011) Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promotors by Eσ70 and EσS, and indirect transcriptional activation by CsrA. Mol. Microbiol. 81:689-704.
- Mukherjee, Sampriti, Helen Yakhnin, Dave Kysela, Josh Sokoloski, Paul Babitzke, and Daniel B. Keanrs. (2011) CsrA-FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis. Mol. Microbiol. (In Press)
- Yakhnin, Helen, Carol S. Baker, Igor Berezin, Michael A. Evangelista, Alisa Rassin, Tony Romeo, and Paul Babitzke. (2011) CsrA represses translation of sdiA, which encodes the N-acylhomoserine-L-lactone receptor of Escherichia coli, by binding exclusively within the coding region of sdiA mRNA. J Bacteriol (In Press)
- Yakhnin, Alexander V., Helen Yakhnin, and Paul Babitzke. (2011) Gel mobility shift assays to detect protein-RNA interactions. Methods Mol. Biol. (In Press)
- Baker, Carol S., Alexander V. Yakhnin, Christopher A. Vakulskas, Igor Berezin, Tony Romeo, and Paul Babitzke. (2011) Molecular mechanism of gene activation by CsrA: Protection of flhDC mRNA from RNase E-mediated cleavage. Proc. Natl. Acad. Sci. USA (Submitted)
- Pannuri, Archana, Helen Yakhnin, Christopher A. Vakulskas, Adrianne N. Edwards, Paul Babitzke, and Tony Romeo (2011) Translational repression of NhaR, a novel pathway for multi-tier regulation of biofilm circuitry by CsrA. J. Bacteriol. (Submitted)
