INHIBITORS OF NUCLEIC ACID SYNTHESIS
CONTENTS CHAPTER 1 THE TRANSITION FROM TRANSCRIPTION INITIATION TO TRANSCRIPTION ELONGATION: START-SITE SELECTION, INITIAL TRANSCRIPTION, AND PROMOTER ESCAPE 1 1 INTRODUCTION 1 2 TRANSCRIPTION START-SITE (TSS) SELECTION 1 2 1 MECHANISM 1 2 2 SEQUENCE DETERMINANTS 1 2 3 BACTERIAL RNAP VS EUKARYOTIC RNAP 1 3 INITIAL TRANSCRIPTION 1 3 1 MECHANISM: ON-PATHWAY REACTIONS 1 3 2 MECHANISM: OFF-PATHWAY REACTIONS 1 3 3 SEQUENCE DETERMINANTS 1 3 4 BACTERIAL RNAP VS EUKARYOTIC RNAP 1 4 PROMOTER ESCAPE 1 4 1 MECHANISM 1 4 2 SEQUENCE DETERMINANTS 1 4 3 BACTERIAL RNAP VS EUKARYOTIC RNAP 1 5 PROSPECTS GLOSSARY ACKNOWLEDGEMENTS REFERENCES CONTENTS CHAPTER 2 MOLECULAR BASIS FOR TRANSCRIPTIONAL FIDELITY CONTROL BY RNA POLYMERASE II 2 1 INTRODUCTION 2 2 STRUCTURAL INSIGHTS INTO TRANSCRIPTIONAL FIDELITY CONTROL BY RNA POLYMERASE II 2 3 CHEMICAL BIOLOGY APPROACHES TO STUDY THE TRANSCRIPTION FIDELITY MECHANISM 2 3 1 DISTINCT CONTRIBUTIONS OF BASE-PAIR HYDROGEN BONDING IN TRANSCRIPTIONAL FIDELITY CONTROL CHECKPOINTS 2 3 2 CRITICAL CONTRIBUTIONS OF SUGAR INTEGRITY IN TRANSCRIPTIONAL FIDELITY CONTROL 2 3 3 ASYMMETRIC DISCRIMINATION OF PHOSPHODIESTER LINKAGE DURING POL II TRANSCRIPTION 2 3 4 USING CHEMICAL BIOLOGY TOOLS TO UNDERSTAND TRIGGER-LOOP RECOGNITION AND CONFORMATIONAL CHANGES 2 4 CONCLUSIONS CHAPTER 3 RNA POLYMERASE AS A TORSIONAL MOTOR 3 1 INTRODUCTION 3 1 1 TRANSCRIPTION GENERATES TORSION 3 1 2 THE BIOLOGICAL IMPLICATIONS OF TORSION 3 1 3 UNDERSTANDING BIOLOGICAL TORSION 3 2 MEASURING THE TORQUE GENERATED BY RNAP 3 2 1 EARLY TORQUE ESTIMATES 3 2 2 THE PATH TO DIRECT TORQUE MEASUREMENTS 3 2 3 THE TORSIONAL PROPERTIES OF DNA 3 3 RNAP'S TORSIONAL PROPERTIES 3 3 1 THE TORQUE GENERATION CAPACITY OF RNAP 3 3 2 OTHER TORSIONAL CHARACTERISTICS OF RNAP 3 4 REGULATION OF RNAP'S TORQUE GENERATION CAPACITY 3 4 1 RNAP'S TORQUE GENERATION CAN BE REGULATED BY A TRANSCRIPTION FACTOR 3 4 2 KINETICS OF RNAP BACKTRACKING AND RESCUE BY A TRANSCRIPTION FACTOR 3 4 3 OTHER FACTORS THAT MAY REGULATE RNAP TORQUE GENERATION CAPACITY 3 5 RNAP TORQUE GENERATION IN CONTEXT 3 5 1 A PHYSIOLOGICALLY RELEVANT TORQUE SCALE 3 5 2 TORQUE- VERSUS FORCE-GENERATION CONTENTS 3 5 3 COMPARISON OF TORQUE GENERATION CAPACITY WITH OTHER MOTOR PROTEINS 3 5 4 FUTURE OUTLOOK CHAPTER 4 RNA POLYMERASE-ASSOCIATED TRANSCRIPTION ELONGATION FACTORS 4 1 INTRODUCTION 4 2 FACTORS ASSOCIATED WITH RNAP DURING TRANSCRIPTION ELONGATION 4 3 ARCHITECTURE OF THE RNAP II ELONGATION COMPLEX – AN OVERVIEW 4 4 1 DISCOVERY AND FUNCTIONS 4 4 2 STRUCTURES 4 5 SPT4–5 AND NUSG 4 5 1 OVERVIEW 4 5 2 SPT4–5 AND NUSG AS PROCESSIVITY FACTORS 4 5 3 OTHER FUNCTIONS OF SPT4–5 AND NUSG 4 5 4 STRUCTURES 4 6 NELF 4 6 1 OVERVIEW 4 6 2 STRUCTURES 4 7 1 OVERVIEW 4 7 2 STRUCTURES 4 7 3 SPT6 IN THE RNAP II ELONGATION COMPLEX 4 8 PAF 4 8 1 OVERVIEW 4 8 2 OTHER FUNCTIONS OF PAF 4 8 3 STRUCTURES 4 9 CONCLUSION CHAPTER 5 RHO TERMINATION FACTOR: ONE RING TO BIND THEM ALL 5 1 INTRODUCTION 5 2 ONE RING TO RULE THEM ALL: STRUCTURE AND DYNAMICS OF RHO ALONE 5 2 1 FUNCTIONAL REGIONS OF RHO 5 2 2 RING DYNAMICS CONTROL RHO MOTOR FUNCTION CONTENTS 5 2 3 THE MECHANISM OF TRANSLOCATION 5 2 4 ALLOSTERIC NETWORKS WITHIN RHO 5 3 THE FELLOWSHIP OF THE RING: CELLULAR PARTNERS OF RHO 5 3 1 NUSG ENABLES RHO TO ACT ON SUBOPTIMAL RNA SEQUENCES 5 3 2 NUSA HAS COMPLEX EFFECTS ON RHO TERMINATION 5 3 3 COOPERATIVE GENE SILENCING BY NAPS AND RHO 5 4 ONE RING TO FIND THEM: ALTERNATIVE MODELS OF RHO RECRUITMENT AND RNA RELEASE 5 4 1 KINETIC COUPLING: AN RNA-CENTRIC, STERIC MODEL 5 4 2 TRAP-AND-RELEASE: AN RNAP-CENTRIC, ALLOSTERIC MODEL 5 5 IT WANTS TO BE FOUND: CATCHING RHO IN THE ACT WITH STRUCTURES OF PRE-TERMINATION ECS 5 6 REGULATING RHO: TIMING ISN'T EVERYTHING 5 6 1 NUSA AND NUSG: DUELING ROLES FOR A DUO OF GENERAL FACTORS 5 6 2 PROTEINS THAT BIND TO AND INHIBIT RHO 5 6 3 SRNAS PROTECT LEADER REGIONS FROM PREMATURE TERMINATION 5 6 4 RIBOSWITCHING OF RHO 5 6 5 CIS-ACTING RNAP APTAMERS 5 7 REGULATORY ROUTES AND CHECKPOINTS IN RHO TERMINATION 5 8 THE IMPORTANCE OF PAUSING IN RHO-DEPENDENT TERMINATION 5 9 R-LOOPS AND GENOME STABILITY 5 10 CONCLUSIONS CHAPTER 6 GLOBAL REGULATION OF TRANSCRIPTION BY NUCLEOTIDES AND (P)PPGPP 6 1 INTRODUCTION 6 2 CHANGING NUCLEOTIDE TRIPHOSPHATE (NTP) CONCENTRATIONS CAN REGULATE TRANSCRIPTION INITIATION AND ELONGATION 6 3 THE NUCLEOTIDE DERIVATIVES PPPGPP AND PPGPP CONTROL TRANSCRIPTION BY MULTIPLE MECHANISMS CONTENTS 6 3 1 HISTORICAL BACKGROUND 6 3 2 REGULATION OF TRANSCRIPTION BY (P)PPGPP BINDING TO ENZYMES RESPONSIBLE FOR THE SYNTHESIS OF NTPS 6 3 3 THE NUCLEOTIDE DERIVATIVES PPPGPP AND PPGPP CONTROL TRANSCRIPTION DIRECTLY BY MULTIPLE MECHANISMS CHAPTER 7 HELICASES AND THE OBSTRUCTIVE RNAP 7 1 INTRODUCTION 7 2 DESTABILIZATION OF TRANSCRIPTION COMPLEXES 7 2 1 UVRD 7 2 2 RHO 7 2 3 MFD 7 3 RNAP AS A LESION SENSOR 7 4 RNAP AS A LESION MEMORY 7 5 MUTAGENIC ACTIVITIES GENERATED BY MOTOR–MOTOR INTERACTION 7 6 CONCLUSIONS CHAPTER 8 TRANSCRIPTION–REPLICATION CONFLICTS, RESOLUTION AND COREGULATION 8 1 INTRODUCTION 8 2 THE DNA REPLICATION COMPLEX 8 3 A HIGHLY DYNAMIC MULTI-PROTEIN COMPLEX 8 4 THE GENE EXPRESSION COMPLEX 8 5 THE CONFLICT: THE TRANSCRIPTION COMPLEX AS A BARRIER FOR REPLICATION FORKS 8 6 THE FORMATION OF R-LOOPS AND THEIR ROLE IN REPLICATION FORK STALLING AND RESTART 8 7 THE ROLE OF TOPOISOMERASES IN AVOIDING CONFLICT AND THE FORMATION OF R-LOOPS 8 8 ADDITIONAL FACTORS REQUIRED FOR CONFLICT RESOLUTION 8 9 DNA DAMAGE AND TRANSCRIPTION ELONGATION 8 10 THE EFFECT OF THE CONFLICT ON GENOME ORGANIZATION AND EVOLUTION CONTENTS CHAPTER 9 USING SINGLE-CELL RNA MEASUREMENTS TO DECIPHER THE STOCHASTIC KINETICS OF TRANSCRIPTION 9 1 PREMISE: “UNDERSTANDING” TRANSCRIPTION 9 2 SINGLE-CELL MEASUREMENTS OF RNA COPY-NUMBER CAN BE USED TO LEARN ABOUT THE STOCHASTIC KINETICS OF TRANSCRIPTION 9 3 CAVEATS 9 4 FROM INDIVIDUAL CELLS TO INDIVIDUAL GENE COPIES 9 5 DETECTING ACTIVE TRANSCRIPTION FROM A SINGLE GENE 9 6 ANALYZING NASCENT MRNA TO REVEAL THE KINETICS OF INITIATION, ELONGATION, AND DEGRADATION 9 7 EVIDENCE FOR PARTIAL RETENTION OF MATURE MRNA AT THE GENE 9 8 A STOCHASTIC KINETIC MODEL CAPTURES SINGLE-CELL STATISTICS 9 9 SISTER GENE COPIES CAN BE HIGHLY CORRELATED IN THEIR TRANSCRIPTIONAL ACTIVITY 9 10 TRANSCRIPTION FROM A REPRESSED PROMOTER IS COUPLED TO THE EVENT OF GENE REPLICATION 9 11 SUMMARY CHAPTER 10 MOLECULAR DYNAMICS SIMULATIONS AND KINETIC NETWORK MODELS ELUCIDATE RNA POLYMERASE TRANSCRIPTION ELONGATION MECHANISMS 10 1 INTRODUCTION 10 2 ATOMISTIC MECHANISM OF THE TRANSCRIPTION ELONGATION CYCLE 10 2 1 STRUCTURAL TRANSITIONS DURING THE TRANSCRIPTION ELONGATION CYCLE 10 2 2 TRANSLOCATION 10 2 3 NTP ENTRY 10 2 4 TRIGGER LOOP CLOSING 10 2 5 NTP ADDITION CATALYSIS 10 2 6 PYROPHOSPHATE RELEASE 10 3 FIDELITY CONTROL 10 3 1 NUCLEOTIDE SELECTIVITY 10 3 2 BACKTRACKING AND INTRINSIC CLEAVAGE 10 3 3 DAMAGED DNA-INDUCED MISINCORPORATION CONTENTS 10 4 CONSTRUCTION OF A KINETIC NETWORK MODEL OF THE ENTIRE TRANSCRIPTION ELONGATION CYCLE 10 5 FUTURE PERSPECTIVES CHAPTER 11 EPILOGUE