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Effects of Local Rna Sequence and Structural Contexts on Ribonuclease P Processing Specificity Jing Zhao Case Western Reserve Un

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Effects of Local Rna Sequence and Structural Contexts on Ribonuclease P Processing Specificity Jing Zhao Case Western Reserve Un EFFECTS OF LOCAL RNA SEQUENCE AND STRUCTURAL CONTEXTS ON RIBONUCLEASE P PROCESSING SPECIFICITY By JING ZHAO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Michael E Harris Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jing Zhao candidate for the degree of Doctor of Philosophy *. Committee Chair Eckhard Jankowsky, Ph.D. Committee Member Michael E Harris, Ph.D. Committee Member Jonatha Gott, Ph.D. Committee Member Derek Taylor, Ph.D. Committee Member Date of Defense March 5th, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein 2 Table of Contents Acknowledgement .......................................................................................................... 8 Abstract ........................................................................................................................ 10 Chapter 1 Background and Significance........................................................................ 13 Background .............................................................................................................. 13 Significance .............................................................................................................. 24 Chapter 2 Distributive enzyme binding controlled by local RNA context results in 3′ to 5′ directional processing of dicistronic tRNA precursors by Escherichia coli ribonuclease P ................................................................................................................................... 27 Abstract .................................................................................................................... 28 Introduction .............................................................................................................. 29 Results ...................................................................................................................... 36 Evidence for 3′ to 5′ ordered processing of dicistronic ptRNAval VW by RNase P in vitro ...................................................................................................................... 36 Kinetic evidence for a distributive processing mechanism ..................................... 46 A stable stem loop in the tRNAvalW 5′ leader inhibits RNase P cleavage and further enforces directional processing.............................................................................. 61 Discussion ................................................................................................................ 65 Materials and Methods .............................................................................................. 73 Chapter 3 ...................................................................................................................... 78 3 Global analysis of sequence specificity landscape for C5 protein using monocistronic ptRNA demonstrates that presence of secondary structure in the leader sequence influences the RNase P processing specificity ............................................................... 78 Abstract .................................................................................................................... 79 Introduction .............................................................................................................. 81 Results and Discussion.............................................................................................. 85 Engineering a stable stem-loop proximal to the P protein binding site reveals the sequence preferences of P protein more clearly ..................................................... 85 The stable stemloop in the 21C distal leader sequence minimizes the effects of unfavorable secondary structure on P protein binding specificity ........................... 89 Quantitative modeling indicates that even in the absense of unfavorable structures within leader sequences, surrounding sequence context is still an important factor for sequence specificity .............................................................................................. 92 The contribution to P protein binding by nucleotides N(-6) to N(-3) depends on the identity of nucleotides N(-2) and N(-1) ................................................................. 98 G in the 5′ leader sequence is an anti-determinant, but only in the presence of a second pairing interaction with the 3′ CCA ..........................................................103 Sequence landscape for RNase P processing specificity .......................................109 Materials and Methods .............................................................................................112 Chapter 4: Summary and Further Directions ................................................................116 Summary .................................................................................................................116 Future directions ......................................................................................................118 4 Appendix .....................................................................................................................121 ....................................................................................................................................148 Reference ....................................................................................................................157 5 List of Figures Figure 1. 1 Recognition model of ptRNA by RNase P holoenzyme ............................... 15 Figure 1. 2 Global precursor tRNA distribution in E.coli K12 MG1655......................... 18 Figure 1. 3 Global secondary structure of precursor tRNA of E.coli K12 MG1655 ........ 20 Figure 1. 4 Proposed free energy landscape of E. coli RNase P ..................................... 23 Figure 2. 1 3′ to 5′ directional RNase P cleavage is the initial step in processing of three dicistronic tRNA precursors 31 Figure 2. 2 In-line structure probing of 5′ 32P end-labeled dicistronic valVW precursor RNA corresponds to predicted secondary structure ....................................................... 39 Figure 2. 3 In vitro processing of of 5′ 32P end-labeled .................................................. 44 Figure 2. 4 Site specificity of RNase P processing of the dicistronic valVW precursor in vitro .............................................................................................................................. 45 Figure 2. 5 Dependence of the observed rate constants and accumulation of reaction intermediate as a function of enzyme concentration ...................................................... 49 Figure 2. 6 Determination of the reversibility of binding at the 5′ tRNA valV processing site ................................................................................................................................ 51 Figure 2. 7 Kinetics analysis RNase P processing of 5′ 32P-labeled dicistronic valWV (A and B), valVV (C and D), and valWW (E and F) substrate RNAs ................................. 56 Figure 2. 8 In-line structure probing of 5′ end labeled dicistronic valWV (A, B), valVV (C, D) and valWW (E, F) substrate RNAs ..................................................................... 60 Figure 2. 9 Single turnover reaction analysis of precursor tRNA rate constants ............. 64 Figure 3. 1 HiTS-KIN substrate pool design and kinetic reaction .................................. 84 Figure 3. 2 Determination of the effects on RNase P processing of 5′ leader sequence variation on the relative kcat/Km for all 4096 substrate variants ...................................... 88 Figure 3. 3 unfavorable secondary structures interfere with C5 protein binding specificity ..................................................................................................................................... 91 Figure 3. 4 PWM modeling of C5 protein binding specificity........................................ 95 Figure 3. 5 Heatmap modeling of C5 protein binding specificity including neighborhood interaction ..................................................................................................................... 97 Figure 3. 6 Sequence variations from N(-6) to N(-3) are interdependent with identity of N(-2) and N(-1)............................................................................................................101 Figure 3. 7 Combination of C(-4)G(-3) is a positive determinant when “A/U” appeared in N(-2)N(-1) positions. ...................................................................................................102 Figure 3. 8 Comparison of the krel value of all 16 nucleotide combinations at N(-4) and N(-2) to the reference sequence ....................................................................................105 Figure 3. 9 variations at the N(-1) position with a “G” at N(-2) indicates that a single base pairing interaction between leader sequence and the 3′ CCA may contribute to krel. .....107 Figure 3. 10 Propopsed Free energy landscape of RNase P processing of substrate pools through multiple-turnover reaction ...............................................................................111 Figure 3. 11 HiTS-KIN processing diagram .................................................................114
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