THE FOCUS OF THIS PROPOSAL IS ON THE EVOLUTION OF PAUSING ONE OF THE REGULATORY STAGES DURING TRANSCRIPTION BY RNA POLYMERASE II (POL II) THAT HAS SO FAR BEEN IDENTIFIED ONLY IN ANIMALS. TRANSCRIPTION IS REGULATED AT EARLY STAGES IN ALL EUKARYOTIC ORGANISMS INCLUDING OPENING CHROMATIN AT THE PROXIMAL PROMOTER AND FORMING THE PRE-INITIATION COMPLEX. IN DROSOPHILA AND MAMMALS POL II PAUSES NEAR THE TRANSCRIPTION START SITE AND AWAITS A SIGNAL TO RELEASE INTO PRODUCTIVE ELONGATION. OUR WORK HAS DEMONSTRATED THAT THE RATE AT WHICH POL II IS RELEASED FROM A PAUSED STATE IS AN ACTIVE TARGET OF GENE REGULATION BY TRANSCRIPTION FACTORS. THEREFORE PAUSING ADDS A LAYER OF REGULATORY COMPLEXITY THAT HAS NOT BEEN REPORTED OUTSIDE OF ANIMAL ORGANISMS. HERE WE PROPOSE TO INVESTIGATE THE EVOLUTION OF PAUSING IN ORGANISMS AT THE BASE OF THE ANIMA PHYLOGENY. WE PRESENT A SPECIFIC HYPOTHESIS FOR HOW PAUSING EVOLVED BY COLLAPSING AN ANCESTRAL STEP NECESSARY FOR TRANSCRIPTION IN ALL EUKARYOTES TO A FOCAL PAUSE ALWAYS OCCURRING IN THE SAME LOCATION ALONG THE DNA SEQUENCE. BY HAVING A FOCAL PAUSE THAT OCCURS IN THE SAME LOCATION TRANSCRIPTION FACTORS WERE ABLE TO EFFICIENTLY CATALYZE THE RELEASE OF PAUSED POL II. THIS ALLOWED RELEASE FROM PAUSE TO BECOME A STEP DURING MRNA PRODUCTION THAT IS ACTIVELY REGULATED BY CELLS. OUR PROPOSAL TO INVESTIGATE THIS HYPOTHESIS WILL PROVIDE MECHANISTIC INSIGHTS INTO HOW PAUSING EVOLVED IN EUKARYOTIC SPECIES REVEALING HOW EVOLUTION ADDED A STEP IN COMPLEXITY TO AN ESSENTIAL BIOLOGICAL PROCESS. OBJECTIVE 1: DETERMINE THE ARCHITECTURE OF TRANSCRIPTIONAL PAUSE IN A DIVERSE GROUP OF ANIMAL AND UNICELLULAR ORGANISMS. WE KNOW VIRTUALLY NOTHING ABOUT PAUSING OUTSIDE OF A FEW MODEL ORGANISMS. HERE OUR GOAL IS TO MEASURE THE DISTRIBUTION OF POL II IN A DIVERSE GROUP OF ORGANISMS USING PRECISION RUN-ON AND SEQUENCING (PRO-SEQ). PAUSED RNA POLYMERASE LEAVES A CHARACTERISTIC PEAK IN THE DISTRIBUTION OF POL II IMMEDIATELY DOWNSTREAM OF THE TRANSCRIPTION START SITE WHICH CAN BE EASILY RECOGNIZED IN PRO-SEQ DATA. PRO-SEQ DATA WILL BE INTEGRATED WITH THE ASSAY FOR TRANSPOSASE ACCESSIBLE CHROMATIN (ATAC-SEQ) TO INVESTIGATE THE INTERDEPENDENCE BETWEEN THE PAUSE AND SURROUNDING NUCLEOSOMES. THIS WILL BE THE FIRST FUNCTIONAL CHARACTERIZATION OF PAUSING IN DEEPLY DIVERGED ORGANISMS AT THE BASE OF THE ANIMAL PHYLOGENY. OBJECTIVE 2: TEST THE HYPOTHESIS THAT PAUSING ALLOWS CELLS TO TAILOR THE RATE OF RELEASE AT SPECIFIC GENES. ALL EUKARYOTIC SPECIES TRANSITION POL II FROM AN INEFFICIENT TO A FULLY TRANSCRIPTIONALLY COMPETENT ELONGATION COMPLEX. OUR HYPOTHESIS IS THAT SPECIES WITHOUT A PAUSE MAKE THIS TRANSITION IN AN APPROXIMATELY UNIFORMLY WAY ACROSS THE GENOME WHEREAS SPECIES WITH A PAUSE TUNE THE RATE OF TRANSITION AT EACH GENE OVER A DYNAMIC RANGE OF>100-1000-FOLD. HERE WE PROPOSE TO TEST THIS PREDICTION DIRECTLY BY USING THE SMALL MOLECULE FLAVOPIRIDOL AND ANALYZING TRANSCRIPTION IN CELLS USING PRO-SEQ. OBJECTIVE 3: TEST THE HYPOTHESIS THAT FOCAL PAUSING IS REQUIRED FOR REGULATION BY TRANSCRIPTION FACTORS IN ANIMALS. WE RECENTLY FOUND THAT HEAT SHOCK FACTOR 1 (HSF1) A TRANSCRIPTION FACTOR THAT IS CRITICAL FOR HEAT STRESS ACTIVATES GENE EXPRESSION BY ORDERS OF MAGNITUDE EXCLUSIVELY BY RELEASING PAUSED POL II. WE WILL USE THIS SYSTEM TO ASK WHETHER HSF1 IS ABLE TO REGULATE GENE EXPRESSION IN A DROSOPHILA MODEL AFTER DEPLETING PAUSED POL II. THIS EXPERIMENT WILL REVEAL WHETHER A FOCAL PAUSE IS NECESSARY FOR ACTIVE REGULATION BY TRANSCRIPTION FACTORS. OUR PROJECT WILL ADVANCE THE GOALS OF THE NASA EXOBIOLOGY PROGRAM BY DETERMINING WHEN PAUSING EVOLVED AND BY PROVIDING THE MECHANISTIC DETAILS OF HOW THIS PROCESS DEVELOPED INTO AN ACTIVE TARGET OF REGULATION OBSERVED IN EXTANT ANIMALS. THUS OUR PROPOSAL ADDRESSES THE BIOLOGICAL FACTORS ESSENTIAL TO MULTICELLULAR LIFE AND THE EVOLUTION OF MULTICELLULARITY ON EARTH.