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Characterization of the Smk Box: a Riboswitch

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Characterization of the Smk Box: a Riboswitch CHARACTERIZATION OF THE SMK BOX: A RIBOSWITCH THAT BINDS S-ADENOSYLMETHIONINE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ryan T. Fuchs, B.S. Graduate Program in Microbiology The Ohio State University 2009 Dissertation Committee: Professor Tina M. Henkin, Adviser Professor Juan D. Alfonzo Professor Charles J. Daniels Professor Kurt Fredrick ABSTRACT Riboswitches are conserved RNA sequences that regulate downstream gene expression via changes in the RNA structure. Riboswitches usually respond to an environmental stimulus, typically binding of an effector molecule, that causes a structural rearrangement without additional trans-acting factors. The structural rearrangement can cause or prevent the formation of an intrinsic transcriptional terminator to regulate the gene at the level of premature transcription termination, or it can modulate the accessibility of the ribosome binding site to regulate at the level of translation initiation. Previous work has shown that there are multiple classes of riboswitches that bind S- adenosylmethionine (SAM) and regulate genes involved in biosynthesis of methionine, cysteine and SAM. The S box (or SAM-I) riboswitches are found in a wide range of bacteria while the SAM-II riboswitch is found primarily in Proteobacteria. Both riboswitches recognize SAM in a similar manner, but they are very different in terms of sequence, secondary and tertiary structure. This demonstrates that it is possible for bacteria to evolve very different regulatory RNAs to respond to the same metabolite. We discovered a third SAM binding riboswitch motif (called the SMK box or SAM-III) upstream of the metK gene (encoding SAM synthetase) in many Lactobacillales species. The SMK box RNA was shown to bind SAM and discriminate against the closely related analog S-adenosylhomocysteine (SAH). Through enzymatic probing, it was determined that SAM causes a structural rearrangement in the RNA that ii results in sequestration of part of the Shine-Dalgarno (SD) region. This observation was confirmed by X-ray crystallography, which also showed that part of the SD directly contacts SAM. A translational fusion of the Enterococcus faecalis metK leader to lacZ was made and introduced into Bacillus subtilis. When the B. subtilis cells were grown under conditions in which SAM pools are elevated, β-galactosidase activity decreased. In contrast, a transcriptional fusion showed no effect. This suggested that the SMK box riboswitch down-regulates gene expression at the level of translation in response to SAM. This observation was supported by the fact that SAM inhibits binding of Escherichia coli 30S subunits to SMK box RNA in vitro. Mutant SMK box sequences that are deficient in SAM binding in vitro showed no SAM dependent effect on 30S subunit binding and no regulation of lacZ expression when SAM pools were modulated. Reverse transcription and quantitative PCR techniques were utilized in order to analyze properties of the metK transcript in vivo in a native organism, E. faecalis. The abundance of metK transcript in E. faecalis cells was found to be unchanged during growth under conditions resulting in high or low SAM pools. This supported the model that regulation of metK by the SMK box is not at the level of transcription. The half-life (t1/2) of the metK transcript was determined to be ~3 min regardless of SAM pools. In contrast, the t1/2 of the interaction between SAM and the SMK box in vitro is 7.8 sec (A. Smith, unpublished results). Thus, the interaction of the SMK box with its effector was shown to be of much shorter duration than the persistence of the transcript in the cell, suggesting that the regulatory effect and structural changes resulting from SAM binding are reversible. iii Attempts were made to demonstrate SMK box-mediated regulation in E. faecalis by utilizing a translational metK-gusA fusion and direct measurement of the activity of the metK gene product, SAM synthetase. Neither method showed regulation of gene expression due to SAM. The basis for these observations is unknown. It is possible that there are other factors involved in regulation of the metK gene in E. faecalis that are masking the effect of the SMK box in vivo. iv ACKNOWLEDGMENTS I wish to express my thanks to my advisor, Dr. Tina Henkin, for her guidance, patience, encouragement, and support throughout the years. It has been a pleasure to work with such a gifted scientist and excellent mentor. I would like to thank Dr. Frank Grundy for all his help and encouragement over the years and for identifying the SMK box motif. I would like to thank my committee members, Dr. Juan Alfonzo, Dr. Charles Daniels, and Dr. Kurt Fredrick for their helpful suggestions, support and time over the years. I also wish to thank all of my colleagues in the Henkin lab for providing a helpful and pleasant working environment. It has been a privilege working with all of you. I would also like to thank my family and friends for support and encouragement throughout my time at OSU. v VITA September 9, 1980……………...Born - Toledo, Ohio June 2002……………………….B.S. Microbiology, The Ohio State University September 2003 – present……...Graduate Research Associate, The Ohio State University PUBLICATIONS Research Publications 1. Fuchs, R.T., Grundy, F.G. and Henkin, T.M. 2006. The SMK box is a new SAM- binding RNA for translational regulation of SAM synthetase. Nat. Struct. Mol. Biol. 13: 226-33. 2. Fuchs, R.T., Grundy, F.G. and Henkin, T.M. 2007. S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA. PNAS 104: 4876-80. 3. Lu, C., Smith, A.M., Fuchs, R.T., Ding, F., Rajashankar, K, Henkin, T.M. and Ke, A. 2008. Crystal structures of the SAM-III/SMK riboswitch reveal the SAM- dependent translation inhibition mechanism. Nat. Struct. Mol. Biol. 15: 1076-83. 4. Fuchs, R.T., Smith, A.M., Grundy, F.J., and Henkin, T.M. 2009. The Enterococcus faecalis SMK box is a reversible riboswitch. (In preparation) FIELDS OF STUDY Major Field: Microbiology vi TABLE OF CONTENTS Page Abstract ..............................................................................................................................ii Acknowledgments...............................................................................................................v Vita ....................................................................................................................................vi List of Tables ......................................................................................................................x List of Figures.....................................................................................................................xi List of Abbreviations........................................................................................................xiii Chapters: 1. Regulation of gene expression by riboswitches…………………………...…..............1 1.1. Riboswitches that respond to tRNA, metal ions, and temperature……………….2 1.1.1. The T box riboswitch...................................................................................2 1.1.2. RNA thermosensors………..……………………………….……………..5 1.1.3. Magnesium-sensing RNAs………………………………………………..7 1.2. The GlcN6P ribozyme……………………………………………………………9 1.3. Metabolite binding riboswitches………………………………………………...11 1.3.1. Riboswitches that bind amino acids……………………………………...11 1.3.1.1.The L box…………………………………………………………….11 1.3.1.2.The glycine riboswitch……………………………………………….13 1.3.2. Adenine and guanine riboswitches……….…………….……………..…15 1.3.3. RFN element………………………………………….………............….18 1.3.4. The Thi box element…..…………………………….…………………...19 1.3.5. The AdoCbl element……………………………………………………..22 1.3.6. Cyclic di-GMP riboswitch.………………………………………………24 1.3.7. PreQ1 riboswitch..……………………………………………………….25 1.3.8. SAH riboswitch……….………………………………………………….27 1.3.9. SAM riboswitches……………….……………………………………….28 1.3.9.1. The S box riboswitch……………………………………..……...28 1.3.9.2. SAM-II riboswitch……………………………………………….32 1.3.9.3. The SMK box riboswitch..……….………………………………..33 1.4. Goals of this study………………………………………………………………35 vii 2. Characterization of the SMK box riboswitch.………..………………………….........36 2.1. Introduction……………………………………..……………………………....36 2.2. Materials and methods…………………………..………………………………38 2.2.1. Generation of DNA templates……………...............................................38 2.2.2. T7 RNAP transcription reactions…………....…………….……………..39 2.2.3. SAM binding assay..……………………………………….…………….40 2.2.4. Structural probing of SMK box RNA with RNase enzymes.……………..40 2.2.5. Bacterial strains and growth conditions………………………………….41 2.2.6. lacZ fusions and β-galactosidase assays…………….…………………...41 2.3. Results……………………………………………………...…………………....42 2.3.1. SMK box RNA binds SAM……………………………………………….42 2.3.2. Mutational analysis of SMK box RNA………..…………………..............43 2.3.3. SAM causes structural changes in SMK box RNA…..…………………...48 2.3.4. Crystal structure of SAM bound SMK box RNA…………………..……..53 2.3.5. Expression of E. faecalis metK-lacZ fusions in Bacillus subtilis…...…...56 2.4. Discussion………………………………………………………..……………...61 3. Analysis of 30S subunit interaction with SMK box RNA……………………..……...65 3.1. Introduction…………………………………………………………..………….65 3.2. Materials and methods……………………………………………..…………....66 3.2.1. Nitrocellulose filter binding assay.………………………………………66 3.2.2. Primer extension inhibition (toeprint) assay………..……………………67 3.2.3. SAM binding assay..…………………………………………………..…67
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