>> GOOD AFTERNOON EVERYBODY. IT'S A PLEASURE TO SEE SO MANY OF YOU HERE FOR THE FALL ANITA B. ROBERTS LECTURE. I'LL THRILLED TO BE HERE AND HAPPY TO HAVE DR. WEI YANG SPEAKING TODAY. I WANTED TO LET YOU KNOW THAT THIS LECTURE SERIES IS SPONSORED BY THE WOMEN'S SCIENTIST ADVISORS OF WHOM I'M ONE. AND ALSO, THE OFFICE OF RESEARCH ON WOMEN'S HEALTH. AND WE ALL FEEL VERY STRONGLY ABOUT PROMOTING WOMEN IN THE INTRAMURAL PROGRAM. THIS IS ONE OF THOSE EFFORTS WE DO THAT. WE RECOGNIZE SENIOR WOMEN SCIENTISTS WHO HAVE MADE OUTSTANDING CONTRIBUTIONS IN THEIR FIELD. THE LECTURE SERIES IS NAMED FOR DR. ANITA B. ROBERTS, AND MANY OF YOU HERE WILL HAVE KNOWN HER. WE'RE VERY HAPPY TO HAVE HER HUSBAND HERE TODAY AS HE'S BEEN FOR ALL BUT ONE LECTURE, I BELIEVE. SO IT'S TERRIFIC THAT HE HAS COME TO HEAR THIS SEMINAR. DR. ROBERTS WAS THE CHIEF OF THE LABORATORY OF CELL REGULATION AND CARCINOGENESIS AT THE NCI. FROM 1995 TO 2006. THEY WORKED ON THE INTRAMURAL PROGRAM FOR 30 YEARS, UP UNTIL HER DEATH. SHE WAS AN OUTSTANDING SCIENTIST, WIDELY RECOGNIZED FOR HER WORK IN CARCINOGENESIS, AUTO IMMUNE DISEASE AND WOUND HEALING AND CONTRIBUTED GREATLY TO OUR UNDERSTANDING OF TRANSFORMING GROWTH FACTOR BETA. IN ADDITION SHE WAS AN EXCELLENT MENTOR AND GREAT AT BALANCING WORK AND FAMILY LIFE, SO FOR WOMEN INCH THE INTRAMURAL PROGRAM, I THINK SHE SERVES AS A WONDERFUL ROLE MODEL FOR ALL OF US ON THE KINDS OF SCIENTISTS AND PEOPLE WE WOULD LIKE TO BE. SO YOU CAN READ MORE ABOUT HER IN YOUR PROGRAMS. AND THE PROGRAMS COME TO YOU COURTESY OF -- WE DID THAT. WSA PEOPLE. SO COURTESY OF US. BUT WE'VE HAD A LOT OF HELP SPONSORING THIS LECTURE. ONE THING I WANT TO POINT OUT IS THE OFFICE OF RESEARCH ON WOMEN'S HEALTH HAD PAID FOR US TO PUBLICIZE THIS SEMINAR SERIES SO POSTERS YOU'VE SEEN AROUND CAMPUS, THE POINTED POSTERS ARE COURTESY OF THEM. IN ADDITION TO THAT, WE -- FOR EVERY SEMINAR WE'VE HAD SO FAR HAS BEEN VIDEO CAST, ALL OF THOSE SEMINARS ARE AVAILABLE ON THE WSA WEBSITE. I ENCOURAGE YOU -- YOU CAN SEE ON THE BACK OF YOUR PROGRAM ALL THE OUTSTANDING WOMEN THAT HAVE SPOKEN IN THIS SERIES. EVERY SINGLE ONE OF THOSE SEMINARS IS AVAILABLE. AND, OF COURSE, YOU -- THOSE IN THE INTRAMURAL PROGRAM KNOW THAT'S A VERY EXPENSIVE PROCEDURE. WE ARE VERY GRATEFUL TODAY TO THE SEARCH COMMITTEE FOR FUNDING THE VIDEO CASTING OF THIS PARTICULAR LECTURE. BECAUSE OF THAT, AND BECAUSE WE ARE VERY IN FAVOR OF PROMOTING AND RECRUITING WOMEN IN THE INTRAMURAL PROGRAM, I WANT TO INTRODUCE ROLAND OWENS, WHO IS GOING TO TALK ABOUT THE TENURE TRACK SEARCH PROGRAM AND ENCOURAGE MORE WOMEN TO APPLY. >> I WANT TO HEAR WEI'S TALK, SO I'LL BE VERY LEAF. I'M HEAR TO ENCOURAGE MORE OF OUR TALENTED WOMEN SCIENTISTS TO APPLY TO THE NIH EARL STATMAN INVESTIGATOR SEARCH. WE KNOW FROM OUR BLINDED FAIR AWARDS COMPETITION, HALF OF OUR TOP POST-DOCS ARE WOMEN. YEAR AFTER YEAR, ONLY ABOUT A THIRD OF THE APPLI CAN'TS TO OUR BROAD ANNUAL SEARCH FOR TENURE TRACK INVESTIGATORS HAVE BEEN WOMEN. WE WANT MORE. IF YOU'RE QUALIFIED TO APPLY, PLEASE APPLY. IF YOU KNOW SOMEBODY WHO SHOULD APPLY, REGARDLESS OF GENDER, PLEASE ENCOURAGE THEM TO APPLY. WE HAVE FLYERS IN THE BACK AND I'LL BE HERE AFTER THE TALK TO TALK WITH ANYONE TO WISHES TO DISCUSS THE SEARCH. THANK YOU. >> WONDERFUL. THANK YOU FOR BEING SO BRIEF. SO NOW IT IS MY PLEASURE TO INTRODUCE DOLLAR WRISTA BUCKNER, SHE WILL INTRODUCE TODAY'S SPEAKER. SHE WAS IN CHARGE OF ORGANIZED THE SEMINAR, SO SHE'S WORKED REALLY HARD FOR THIS. >> THANK YOU. OUR SPEAKER TODAY DR. WEI YANG EXOPLASMIFYIFIES MANY OF THE SAME CHARACTERISTICS THAT MADE DR. ROBERTS SUCH A LOVED AND RESPECTED MEMBER OF THE NIH COMMUNITY. RECENT ELECTED TO THE NATIONAL ACADEMY OF SCIENTISTS DR. YANG IS A PASSIONATE ACCOMPLISHED SCIENTIST. HER SCIENTIFIC CAREER STARTED WITH A B.A. FROM THE STATE UNIVERSITY OF NEW YORK AT STONEY BROOK. FOLLOWED BY A Ph.D. FROM COLUMBIA UNIVERSITY. DR. YANG PERFORMED HER POSTDOCTORAL TRAINING AT YALE UNIVERSITY. IN 1995 SHE CAME TO THE NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES AS A TENURE TRACK SENIOR AND STAFF FELLOW. NOW, A TENURE SENIOR INVESTIGATOR AND SECTION CHIEF OF THE LABORATORY OF MOLECULAR BIOLOGY IN NIDDK, DR.IAC USES X-RAY AND OTHER METHODS TO STUDY THE ENZYMES INVOLVED IN DNA REPAIR, RECOMWITHIN NATION AND REPLICATION. SHE HAS A TRACK RECORD OF BEING SCIENTIFICALLY AHEAD OF HER TIME AND PERSISTENT. DURING HER POSTDOCTORAL TRAINING, SHE WANTED TO STUDY THE MECHANISMS OF RECOMBINATION ACTIVATEING GENE RECOMBINATION. THE PROCESS BY WHICH ANTIBODIES AND RECEPTORS ARE ASSEMBLED IN IMMUNE CELLS. HOWEVER, TOOLS NEEDED FOR SUCH STUDIES WERE UNDERDEVELOPED. SHE FOCUSED ON GAMMA DELTA, AND ENZYME INVOLVED. HER DESIRE TO WORK ON VDJ RECOMBINATION NEVER FADED, AND WAS REVISITED AT THE NIH WITH THE COLLABORATION OF ANOTHER DOCTOR. IT ALSO WAS HERE AT THE NIH THAT DR. YANG IDENTIFIED UNEXPECTED ENZYMATIC ACTIVITY IN ONE OF THE PROTEINS THAT PLAYS AN IMPORTANT ROLE MAINTAINING STABILITY. HER DISCOVERY WAS CONTROVERSIAL AND TOOK SOME TIME TO PUBLISH. BECAUSE OTHER SCIENTISTS ATTRIBUTED HER FINDINGS TO CONTAMINATION DURING PROTEIN PURIFICATION PROCEDURES. THIS GROUNDBREAKING WORK HAS BEEN CITED BY OVER 75 ARTICLES. RECENTLY, DR. YANG AS HER TEAM DEMONSTRATED HOW 3D STRUCTURES MACRO MOLECULAR COMPLEXES PROVIDE UNIQUE INSIGHTS INTO DYNAMIC INTERACTIONS BETWEEN PROTEINS AND DNA IN HEALTH AND DISEASE. COLLABORATIONS WITH OTHER INVESTIGATORS HERE AT THE NIH HAS LED HER TO THE FIELD OF HIV. THEIR STRUCTURAL WORK PROVIDES INSIGHTS INTO THE MOLECULAR MECHANISM FOR HIV RESISTANCE TO A PROCEED RANGE OF ANTI-RETROVIRALAL DRUGS. THIS WORK IS LEAD TO THE DEVELOPMENT OF NOVEL ANTI-HIV THERAPEUTICS. IN ADDITION TO BEING AN ELECTED MEMBER OF THE NATIONAL ACADEMY OF SCIENCES, DR. YANG IS A RECIPIENT OF SEVERAL AWARDS. SHE HAS PUBLISHED OVER 80 ORIGINAL RESEARCH PAPERS AND 3 BOOK CHAPTERS. FINALLY, IN ADDITION TO HER SCIENTIFIC ACHIEVEMENTS, DR. YANG IS ALSO A GREAT MENTOR, ENCOURAGING AND PROMOTING THE EDUCATION AND CAREER DEVELOPMENT OF ALL OF THE STUDENTS AND FELLOWS IN HER LABORATORY. SHE HAS MENTORED OVER 30 POST BACK LORETTE GRADUATE AND POSTDOCTORAL FELLOWS. SHE IS VERY PROUD OF THE FELLOWS SHE HAS TRAINED, THE MAJORITIES OF WHICH HAVE BECOME PROFESSORS. WITH A MANTRA OF WHEN WORK WITH ONE'S HOBBY AND PASSION, CREATIVITY COMES NATURALLY. IT'S NO WONDER SHE HAS HAD SUCH A DISTINGUISHED CAREER. WITHOUT FURTHER ADO, I PRESENT TO YOU DR. WEI YANG. [APPLAUSE] >> THANK YOU VERY MUCH. MAKES ME RED. SO IT'S MY GREAT HONOR TO SPEAK HERE AT THE ANITA B. ROBERTS LECTURE SERIES AND JOINING THE DISTINGUISHED WOMEN'S SCIENTISTS AT NIH IN THE U.S. THANK YOU FOR SELECTING ME AND THE OPPORTUNITY. MY FIRST ENCOUNTER WITH ANITA WAS WALKING INTO THE VERY INVITING GARDEN WITH A CHARMING FROG DEDICATED TO HER MEMORY. IN FRONT OF BUILDING 37. AND I DIDN'T KNOW WHO IS ANITA B. ROBERTS. THEN LOOKING TO HER LIFE AND WORK AND I HAVE TO SAY I'M IN AWE OF HER ACCOMPLISHMENT AND AMAZING LEGACY AND HUMBLED TO BE ASSOCIATED WITH THIS NAME TODAY AND THE GROUP ON SELECTING ME. SO WHAT I DECIDED TO DO TODAY IS SHARE WITH YOU A 25 YEAR JOURNEY OF STUDYING REVERSE TRANSCRIPTASE ENCODED BY A RETROVIRUS. AND BECAUSE IT'S INTERESTING, PERSISTENCE, ALONG THE WAY, I HAVE RECEIVED MANY HELP, MUCH HELP FROM MANY, MANY COLLEAGUES, COLLABORATORS INSPIRED BY FELLOWS AND STUDENTS WITH THEIR DRIVE AND CREATIVITY. SO REVERES TRANSCRIPTION IS A PROCESS THAT CONVERTS GENOME RNA TO DOUBLE STRANDED DNA TO BE INTEGRATED INTO A HOST CHROMOSOME. IN THE PROCESS, CATILOUSED BY A SINGLE ENZYME CALLED REVERSE TRANSCRIPTASE, WHICH CONTAIN A DNA POLYMERASE THAT SYNTHESIZE THE DNA USE, READ AS A TEMPLATE TO MAKE DOUBLE STRANDED RNA. THE ACTIVITY IS ESSENTIAL TO REMOVE THE TEMPLATE RNA WHEN IT FORMS A HYBRID WITH DNA. AND H STANDS FOR HYBRID, BECAUSE RNA ONLY WHEN IT'S IN THE RNA DNA HYBRID. IT'S ESSENTIAL TO REMOVE THE TEMPLATE TO COMPLETE THE PROCESS, AND ALSO, TO GENERATE POLY -- A SHORT STRETCH OF RNA TO PRIME SECOND STRAND OF DNA SYNTHESIS. AND NEEDLESS TO SAY THAT HIV REVERSE TRANSCRIPTASE IS EXTENSIVELY STUDIED IN THE LAST 20 ALSO YEARS. AND THERE ARE HUNDREDS OF STRUCTURES OF HIV RT.ED IN THE DATA BANK. ALL LOOSE STRUCTURESKY BE SUMEROUSED IN TWO STATE. ONE STATE IS WHEN THE INSOMETIME BINDS WITH DNA SUBSTRATE SHOWING DOUBLE HELP LINKS WITH AN INCOMING NUCLEOTIDE WHICH CAN BE MIMMICKED BY A NUCLEOTIDE INHIBITOR, LIKE, SHOWN HERE. AND A CLOSE CONFIRMATION. THE OTHER CONFIRMATION OF STATE IS REPRESENTED BY I.T. BOUND TO A NON NUKE CLEO TIDE INHIBITOR. IN THE OPEN STATE. IF I REMOVE THE DNA DUPLEX, YOU WILL SEE THE INHIBITOR BINDING SITE, THE ACTIVE SITE OF POLYMERASE MUCH BETTER. NOW THE INHIBITOR IN R.T.I. IS SHOWN HERE, AND IT'S FOUND BETWEEN THE GREEN DOMAIN AND RED SUBDOMAIN. BY BINDING, STABILIZED AN OPEN CONFIRMATION OF THE R.T. PREVENTED INTO THE MODE OF SYNTHESIZED DNA. THOSE TWO R.T. INHIBITORS HAVE BEEN DEVELOPED IN MANY DIFFERENT FORMS, AND HAS BEEN USED EXTENSIVELY IN TREATMENT OF HIV PATIENTS. HOWEVER, MUTATIONS LEADS TO DRUG RESISTANT OVER THE YEARS. AND NO MATTER HOW ONE CHANGES THE DRUG, THE BINDING POCKET IN THE PROLIMERASE DOESN'T CHANGE. THE RNA ACTIVE SITE IS RESIDED HERE, QUITE A BIT OF -- AWAY FROM THE POLYMERASE. IS THE ONLY ONE SO FAR, NOT AS A DRUG TARGET TO TREAT HIV. SO THAT WAS WHERE WE WANTED TO FOCUS OUR CHALLENGES. AND I START MY JOURNEY OF STUDY, REVERSE TRANSCRIPTASE IN 1986 WHEN I BECAME AFRAID STUDENT OF WAYNE HENDRICSON AT COLUMBIA UNIVERSITY. AND JUST AT THAT TIME, STEVE GOLF'S LAB CLONED ANOTHER RETROVIRAL TRANSCRIPTASE, AND THE POSTDOC MONICA ROTH, NOW A PROFESSOR AT RUTGERS UNIVERSITY, DEVELOPED OLD EXPRESSION SYSTEM AND PURIFIED R.T. SO THE COLLABORATION BETWEEN WAYNE, AND -- LED ME TO WORK WITH MONICA ACTION AND SHE TAUGHT ME HOW TO PURIFY A PROTEIN. THE WORK WAS NOT COMPLETE UNTIL BOB CROUCH, A SCIENTIST FROM NICHD, JOINED THE TEAM. AND WAS A MEMBER OF MY COMMITTEE AND HIV I.T. AS I SHOWED YOU BEFORE, IT WAS A FAIRLY LARGE ENZYME, CONTAINED OF TWO POLY PEP TIDE CHAINS, POLYMERASE RESIDES ON THE SAME CHAIN. THE VIRUS, WHICH MONICA ROTH WORKED ON WITH STEVE GOLF WAS SIMPLIST, ONLY ONE CHAIN. AND IN 198786, WE THOUGHT -- WORKING ON A SINGLE POLY PEPTIDE ENZYME, WORKING ON LESS THAN AGGRESSIVE VIRUS, MAYBE IT'S -- AN EASY WAY TO, YOU KNOW, SOLVE THE STRUCTURE OF REVERSE TRANSCRIPTASE. BUT THINGS TURNED OUT NOT WHAT WE IMAGINED. AND TURNED OUT THAT THE VIRUS JUST VISIT THE STRUCTURE OF DETERMINATION. THE FIRST STRUCTURE OF DOMAIN OF MUR REASON R.T. CAME OUT IN 1995. TEN YEARS LATER. SO AFTER SPENDING TWO YEARS WORKING ON LEUKEMIA VIRUS R.T. FOR TWO YEARS, NOT GETTING VERY FAR ALONG, I WAS DEPRESSED AND MONICA WAS URGING ME ON. THE ONE DAY, WAYNE SAID MAYBE WE SHOULD SCALE BACK OUR TARGET. MAYBE WE SHOULD WORK ON THE RNA PHAGE, WHICH HAVE A REVERSE TRANSCRIPTASE, E. COLI ENCODE ITS ONLY SELLIER PHASE. MUCH SMALLER AMINO ACID AND SIMPLER. YOU WILL HEAR SOON WHY IT'S IMPORTANT. SO SOON, BOB CROUCH, BOB WAS THE ONE DISCOVERED IT IN THE FIRST PLACE, HE WAS VERY HELPFUL, SENT US THE PURIFIED PROTEIN, THE CLONE, AND INVITED ME DOWN HERE IN NIH IN 1988 TO WORK WITH HIM TO LEARN HOW TO PURIFY THE PROTEIN. THAT INITIATES AND LAURENED THE PROGRESS TO SUCCESS IN MY Ph.D. DEGREE. THE WAY INTERESTED IN USING THE [INDISCERNIBLE] TO REPLACE IT PAUSE IN CRYSTALLOGRAPHY, TO GET FACE, [INDISCERNIBLE], IT WAS VERY TRIAL AND ERROR. A LOT OF EFFORT IS PUT IN. ONE NEVER KNOWS WHETHER IT WILL WORK. BY REPLACING IT, AND USE THE METHOD CALLED MAD, NOW IT'S RELIABLE. ONCE YOU GET CRYSTAL, IT'S RELIABLE WAY TO SOLVE THE STRUCTURE TO DETERMINE -- USING THIS SE MET METHOD. NOW DAYS THIS ENABLED THE STRUCTURED GENOMICS TO TAKE PLACE AND ABOUT 50% OR MORE OF THE STRUCTURES IS USING THE SE MET REPLACEMENT. SO IT WAS THE FIRST STRUCTURE USING THIS METHOD UNDER WAYNE'S GUYEDANCE. HERE ARE THE TWO. THE RED DOTS YOU SEE IS WHERE THE SELENIUM FITS, VERY HIGH DENSITY AND INTERESTING SCATTERING PROPERTIES, CARBON, NITROGEN, OXYGEN OF THE NORMAL PROTEIN COMPOSITION. SO WHEN WE DETERMINE RNA, THAT WAS IN 1990, THEN THECO WAS NOT LIKE TODAY. AND THE PICTURE OF HOW THE PROTEIN LOOK LIKE WAS NOT GENERATED BY COMPUTER PROGRAMS. THIS IS HAND DRAWN BY AN ARTIST. AND THE PROTEIN REPRESENTS A NOVEL FOLD AND THE STRUCTURE, WE WERE ABLE TO IDENTIFY ACTIVE -- FOUR VERY CONSERVED [INDISCERNIBLE] THAT DEFINES THE ACTIVE SITE. NOT ONLY THAT WE FOUND ACTIVE SITE, WE WERE ABLE AND VERY PROUD OF LOOKING BACK IN 1990, WE WERE ABLE TO PROPOSE HOW THE RNA DNA HYBRID MAY BIND TO RNA. THE BIG RED DOT IS THE ACTIVE SITE, REPRESENTS CHARGE. AND THE BLUE DOTS SURFACE REPRESENTS POSITIVE CHARGE WHICH IS ATTRACTED TO THE BACK BONE OF DNA OR RNA. AND WHAT'S INTERESTING WHEN WE CALCULATE THE SURFACE POTENTIAL, WE NOTICE THE ACTIVE SITE IS QUIT CONNECTIVE, WHICH REQUIRES [INDISCERNIBLE] TO NEUTRALIZE IT IN ORDER FOR IT TO BIND SUBSTRATE. AND THE VERY STRONG PHOSPHATE BINDING POCKET, WHICH IS OCCUPIED BY A PHOSPHATE, IS QUITE THE DISTANCE AWAY. THAT CANNOT BE ON THE SAME STRAND, SO WE HYPOTHESIS THAT BY LOOKING THE DENT, THAT THE RNA STRAND GOING THROUGH THE ACTIVE SITE BEING CLEAVED. THE DNA STRAND GOING DOWN THROUGH THE PHOSPHATE BINDING POCKET IN THE GROOVE. AND THAT WAS THEN, IN 1990. AND FOR THE NEXT 12 YEARS, I KNOW THAT DR. HENDRICSON'S GROUP WAS WORKING ON E. COLI RNA AND COMPLEX WITH DNA HYBRID. FOR SOME REASON THE STRUCTURE NEVER MATERIALIZED, BECAUSE SEEMS TO BE THE ENZYME LIKE TO BIND THE SUBSTRATE AND CULT IT AND RELEASE IMMEDIATELY. IT WOULDN'T SAY THERE TO BE TRAPPED IN THE ENZYME SUBSTRATE STATED. MINE WHILE RNA, WHEN WE SOLVED THE STRUCTURE, IT LOOKS LIKE A NOVEL FOLD. IN THE NEXT 10, 15 YEARS, IT POPPED UP EVERYWHERE. INCLUDING THE HIV INTEGRACE WHICH UTILIZED THE PROTECT OF REVERSE TRANSCRIPTION, AND VIRAL DNA INTO THE HOST CHROMOSOME. THE INTEGRACE CONTAINED DOMAIN. SO MANY DNA -- AND MORE RECENTLY THE FAMOUS PROTEIN IN RNA, RNA I INTERFERING PATHWAY ALSO CONTAIN RNA DOMAIN. THAT ADDRESSED THE QUESTION, HOW RNASE REAL BIND THE SUBSTRATE. FAST FORWARD. IN 19 -- IN 2002, IN -- BY THEN, I HAD ON THIS CAMPUS FOR SEVEN YEARS. BOB AND I MET EACH OTHER EVERY ONCE IN A WHILE TO TALK ABOUT THE GOOD OLD DAYS. THEN HE SAID WHY DON'T WE START TEAM UP, LOOKING AT THE COMPLEX STRUCTURE IN THE SUBSTRATE? AND I WAS PEEKED. AT THAT TIME, LUCKY, VERY MOTIVATED AND VERY TALENTED POST-DOC MARCIN JOINED MY GROUP. WE STARTED OUR JOURNEY AGAIN. THE TEAM WAS BOB'S POST-DOC, SERGEY. CLONED 6 DIFFERENT RNA'S FROM DIFFERENT SPECIES. LOOKING FOR ONE THAT WOULD BE SO GOOD IT WILL BIND THE SUBSTRATE, BUT NOT CLEAVING IT TOO WELL. AND SO HE SEARCHED FOR THAT. AND IN CONJUNCTION THAT WE KNOW WITH ACTIVE SIGHT LEAKED LIKE HE MADE THE MUTATIONS TO MUTATE ESSENTIAL CAR BOX LATE. SO IT HELPED HIM TO SCREEN FOR THIS PROTEIN TO SEE IF THEY BIND THE RNA DNA HYBRID BETTER. THIS WAS WILDTYPE DURING RNA. THIS IS THE SUBSTRATE, THE PROTEIN, THE DNA COMPLEX. IT WAS INCREASING AMONG THE PROTEIN. AND THE MUTANT, SHOWED IT BINDS MUCH PETER AT LOWER CONCENTRATION WITH PROTEIN, ALREADY FULLY FORM ADD COMPLEX, GIVES A WAY TO GET THE STRUCTURE DETERMINED. AND I'M HAPPY TO REPORT THAT THE STRUCTURE OF HAL OUTSTANDING DURAN RNASE, IS AN APPLICATION AND THE STRUCTURE WAS DETERMINED BECAUSE THE ACTIVE SITE CAR BOX LATE, WE CAN PUT IN SUBSTRATE AND THE METAL MAGNESIUM, THE TWO YELLOW DOTS ON THE SURFACE OF PROTEIN MAGNESIUM IONS. YOU CAN SEE THE TWO GROUPS AS WE PROPOSED EARLIER. IT'S RNA STRAND GOES THROUGH THE ACTIVE SITE, AND THE DNA STRAND IN BLUE GOES THROUGH THIS LOWER GROVE, WHERE THE PHOSPHORYLATE BINDING POCKET WAS VERY OBVIOUS, EVEN IN THE E. COLI STRUCTURE. THIS BINDING POCKET SORT OF TUCKED THE DNA STRAND, WAS THE PROTEIN AND THEY REQUIRED THIS TO BE ACTUALLY DNA. AND RNA RESIDUE WOULD NOT BE ABLE TO FIT INTO THIS POCKET. SO TO ENSURE THAT ENZYME WILL RECOGNIZE RNA DNA HYBRID, AND THE CLEAVE RNA STRAND ONLY. AND WENT ON TO DETERMINE THE STRUCTURE OF RNASE AND ALSO REACTION TO ILLUSTRATE HOW THE REACTION ACTUALLY GOES. IN THE SUBSTRATE STATE, THESE ARE THE TWO MAGNESIUM I DIDN'T ONS. MAGNESIUM LIKES TO HAVE 6 LIGAND COORDINATING MAGNESIUM. IN THE SUBSTRATE STATE, THERE IS ONLY 5 LIGANDS. IT'S NOT VERY STABLE. SO WANT TO GET OUT OF THE SITUATION AND WE INTERPRET THAT, THAT'S HOW IT'S ACTIVATING, ATTACKING WATER. THE MAGNESIUM HELPING TO ACTIVATE THE WATER FOR IT TO ATTACK -- FROM SUBSTRATE TRANSITION INTO IMMEDIATE. AND WE OBVIOUSLY KNOW THE REAL STRUCTURE. THIS IS HIPSSIS. WHEN THE REACTION IS COMPLETE THE BOND IS CLEAVED AND THE PHOSPHATE IS RELEASED AWAY. YOU CAN SEE THAT, ONE OF THE METAL ION IS ONLY COORDINATED BY FIVE LIGANDS, NOT VERY STABLE. IN THE PRODUCT STATE, WHEN THE BOND IS CLEAVED AND THE TENSION IS RELEASED, THIS BECOMES 6 LIGAND. AND IN A RELAXED STATE. YOU SEE THE METAL ION MOVE APART. IN FACT, WHEN IT WAS SHOWED HOW THE REACTION OCCURRED IN RNASE, THE LAB DETERMINED TWO OTHER STRUCTURES, NUCLEAR CLINIC ACID ENZYMES ALL DEPENDED ON TWO IONS, AND INTERESTINGLY, WE LINE UP THE TOP ROW OR THE POLYMERASE WHICH CONTAINED REVERSE TRANSCRIPTASE. THE BOTTOM ROW ARE NUCLEAR CLINIC ACID ENZYMES THAT CLEAVE NUCLEARLIC ACID, ALSO RIBOZYME, MADE UP OF RNA, AND SELF-CLEAVE IT'S ON RNA. THE OTHER STRUCTURES, TWO GREEN ATMOSPHERE REPRESENTING METAL IRON, VERY IMPORTANT. THE BINDING MODE ARE VERY SIMILAR. THAT PROPPED US TO THINK, WHY NUTTRIC ACID ENZYME? AND WHY NOT USE PROTEIN? AND WE HYPOTHESISED. YOU CAN HAVE DIFFERENT ANGLE, DIFFERENT DISTANCE, MAGNESIUM IS STRINGENT, ANGLE VERY DEFINED. IF THE BINDING ENVIRONMENT IS NOT SATISFIED, THE REACTION WOULDN'T OCCUR. MAGNESIUM WOULDN'T BIND. THAT EXPLAINS WHY MANY ENZYMES, FOR EXAMPLE, CAN BIND VARIOUS DNA SITES BUT THE CLEAVAGE ONLY OCCURS WITH THE SEQUENCE, AND THE MISSING LITTLE BIT OF BASE PAIR PERFECTION, THEN THE MAG SEIUM WOULDN'T BIND. SO MAGNESIUM ENSURES THE SPECIFICITY OF NUCLEARLIC ACID ENZYME. ONLY FOUR BASES, OVERALL STRUCTURE IS VERY SIMILAR. YOU WANT IT TO CLEAVE AT A PARTICULAR PLACE AT A PARTICULAR TIME. TO FURTHER DEMONSTRATE HOW THE MAGNESIUM WORKS, I WILL SHOW YOU THE RECENT WORK A JOY TO SHARE WITH YOU, THAT WE WERE ABLE TO WATCH CHEMICAL REACTION CATALYZED BY AN ENZYME. THIS WORK WAS STARTED WITH A COLLABORATION SOMEONE IN JAPAN, A POST-DOC AND STUDENT, NOW ALL P.I.'S IN THEIR COUNTRY AND CONTINUING BY A GRADUATE STUDENT HERE IN A JOINED BY VISITING SCIENTIST CAME FROM JAPAN FOR A FEW MONTHS. SO THE WAY WE LOOK AT POLYMERASE DATA, THE REASON WE CHOSE IT, THE ONLY KNOWN HUMAN POLYMERASE. IT HAS A CANCER PHENOTYPE. THE DNA IS ESSENTIAL FOR TRANSLATION BYPASS OF UV LESION. WHEN WE GO OUT IN THE SUN, OUR DNA ARE DAMAGED TO ADJACENT, CAN BE CROSS LINKED BY UV LIGHT. THAT BLOCKS THE REGULAR REPLICATION BY POLYMERASE INCLUDING REVERSE TRANSCRIPTASE. AND PEOPLE WITH INACTIVE -- INACTIVATEED [INDISCERNIBLE] POINT MUTATIONS, THEY HAVE SENSITIVITY TO UV LIGHT AND DEVELOP SKIN LESIONS. POLH. IT'S ABLE TO INSERT CORRECTLY THE A BASE OPPOSITE DIMER. WITH THREE YEARS HARD WORK WE EVENTUALLY SOLVED THE STRUCTURE COMPLEX WITH DNA CONTAINING THE LESION. THIS TWO BASE LINKED SHOWING ORANGE COLOR, THAT IS CAUSED BY UV. AND IT'S ABLE TO INSERT CORRECTLY THE D.A.T.P. HYDROGEN BOND TO THE CROSS LINK BASE. THE REASON ACTIVE SITE IS LARGE AND CARVED OUT TO SPECIFICALLY ACCOMMODATE THIS LESION. PLAYING A ROLE IN CHEMO RESISTANCE TO TREATMENT. CROSS LINKING TWO BASES, MIMICKING THE STRUCTURE OF UV CROSSED BASE. SO IN A WAY, IT'S ESSENTIAL FOR US TO SURVIVE THE UV LESION. ON THE OTHER HAND, IT'S A DRUG TARGET TO ENHANCE THE TREATMENT FOR CANCER PATIENTS. IN THIS SIDE, WE WILL FOCUS ON THE ACTIVE CENTER OF IT. REGARDLESS WHAT KIND OF POLYMERASE IT IS [TECHNICAL DIFFICULTIES] THE REASON WE WERE ABLE TO GET THIS CRYSTAL WAS A LOT OF HARD WORK BY CHRISTIAN AND SPEND 3 YEARS ENGINEERING THE PROTEIN, THE DNA, TO CRYSTALLIZE THE COMPLEX. IN THE PERFECTLY ACTIVE FORM. SO YOU SEE THE PERFECT ALIGNMENT AND GOOD DISTANCE. THE REASON WE WERE ABLE TO TRAP ENZYME SUBSTRATE COMPLEXAA WAS BECAUSE WE USED A NON REACTIVE SUBSTRATE, REPLACING OXYGEN, WHICH SHOULD BE READ HERE WITH NITROGEN. SO BECAUSE THIS BOND CANNOT BE BROKE, SO THIS BOND CANNOT FORM. AND THAT'S WHAT CRYSTAL GRAPHERS HAVE BEEN DOING FOR MANY, MANY DECADES, BASICALLY LOOK AT ENZYME SUBSTRATE COMPLEX, PREVENTING THE REACTION AND TAKING A STATIC IMAGE. NO ONE HAS LOOKED AT THE REACTION PROCESS ITSELF BECAUSE YOU CANNOT TRAP IT. GIVEN THAT CRYSTAL IS SO PERFECTLY ALIGNED, WE FIGURE OUT A WAY TO LOOK AT REACTION IN REAL TIME, WITH REAL SUBSTRATE. THE PROCESS IS THAT WE TAKE IT ADVANTAGE THAT THE REACTION IS PH DEPENDENT, LOW PH, REACTION DOESN'T OCCUR. THE SECOND REQUIREMENT IS TWO MAG NIECIUM TO ALIGN THE SUBSTRATE. WE CRYSTALIZE THE PERFECT ENZYME, IN THE PRESENCE OF ONE CALCIUM PER COMPLEX. IT ALLOWED THE COMPLEX TO FORM, NO REACTION CAN OCCUR. THEN THE PH WAS RAISED TO THE REACTION RANGE, 6.8 TO 7.2. AND WE WERE LUCKY THAT CRYSTAL DIDN'T DISSOLVE AND RETAINED THE DEFRACTION POWER TO 1.5. THEN THE CRYSTAL WAS DIPPED INTO MAGNESIUM. REACTION STARTED TO OCCUR. FORTUNATELY, THE REACTION CRYSTAL WAS VERY SLOW, ENABLING THE CRYSTAL TO BE FREEZED AT VARIOUS TIME INTERVALS. 40 SECOND, 80, 120. BY FREEZING THE COMPLEX, WE CAN FREEZE THE REACTION. THEN WE CAN TAKE X-RAY DEFECTION TO FOLLOW THE REACTION WITH THOSE TIME POINTS. AND THAT'S IN THE FOLLOWING SHIED, I WILL SHOW YOU THE TIME LAPSE OF THE REAL REACTION, VERY HIGH RESOLUTION. AT THE 0 TIME POINT WHEN WE ONLY PROVIDE ONE CALCIUM, YOU CAN SEE IT'S VERY IMPORTANT TO NEUTRALIZE THE CONNECTIVE CHARGE OF THE CATALYTIC CAR BOX LATE AND THE PHOSPHATE IN THE INCOMING NUCLEOTIME. WITHOUT IT, THE COMPLEX WOULDN'T FORM. WITH THE SECOND METAL WHAT YOU SEE IS THAT THE [INDISCERNIBLE] IS NOT PERFECTLY ALIGNED FOR ATTACKING PHOSPHATE. HERE IS THE INCOMING NUCLEOTIDE. THE TEMPLATE DNA IS IN THE BACK. YOU WOULDN'T SEE IT. YOU SEE A CONCERNED [INDISCERNIBLE] THAT PLAY A VERY NICE ROLE, SORT OF FORK STABLE THE BETA PHOSPHATE. IN SHORT, CALCIUM ENABLED BINDING OF SUBSTRATE TOGETHER, BUT WITHOUT ALIGNING THE REACTANT. THEN WE SOAKED THE CRYSTAL IN MAGNESIUM. NOW, THE TWO MAGNESIUM OCCUPY THEIR PROPER POSITION, BECAUSE THE BASE PAIR IS PERFECT. WE ANTICIPATE IF THE BASE PAIR IS NOT CORRECT, THE SITE WOULDN'T FORM BECAUSE THE MISALIGNMENT OF THE LIGAND. SO NOW IN THE PRESENCE OF TWO MAGNESIUM, WHAT YOU SEE IS THAT THE PRIMER STRAND IS PERFECTLY ALIGNED WITH [INDISCERNIBLE] FOR REACTION. IF YOU'RE NOT CONVINCED I'LL TOGGLE THE PICTURE WITH ONE CALCIUM, WITH TWO MAGNESIUM. AND IT'S CLEARLY SHOWS WHAT ROLE THE MAGNESIUM SERVES. ALIGNING THE REACTANT. BUT EVEN WHEN THE REACTANT ARE PERFECTLY ALIGNED AT 40 SECONDS, WE DIDN'T SEE THE REACTION TAKE PLACE. SUGGESTING THERE IS A VERY LIMITING STEP FOR THE REACTION TO TAKE PLACE. SO THE IN CRYSTAL TIME LAPSE STUDY ALLOWS TO WATCH WHAT HAPPENED TRANSIENTLY. AND WE ARE MONITORING THE BOUND FORMATION HERE, INDICATED BY A CLOUD OF ELECTRON DENSITY SHOWING UP. HERE IS ZOOMING TO THE ACTIVE CENTER. THIS IS THE TIME SERIES, 140, 230 SECONDS. ONE VIEW, VERY SIMILAR TO WHAT YOU SAW ALREADY. THE BOTTOM ROW IS A DIFFERENT VIEW OF THE SAME TOMORROW POINT LOOKING FROM TOP DOWN. SO YOU SEE A DIFFERENT ARRANGEMENT. 40 SECONDS DOES NOTHING. 80 SECONDS, THEY START TO MERGENED START TO FORM AND THE BOND INTENSITY INCREASES. WE CAN MONITOR THE CHEMICAL REACTION. WHAT'S INTERESTING IS THAT BETWEEN 40 AND 80 SECONDS WE SEE A BIG CHANGE IN ELECTRONIC OF ELECTRON DENSITY. A DENSITY WASN'T SHOWING IN THE 40 SECOND, A-- 80 SECONDS, AND THEN IT DISAPOWERED WHEN THE REACTION GOES TO COMPLETION. AND WE ASSIGN THIS AT THE WATER MOLECULE, HIGH TROJAN BOUND WHICH NEED TO BE INITIATE THE REACTION. THE FIRST TRANSIENT ELEMENT WE FOUND. SECOND TRANSIENT ELEMENT ONLY SHOWS UP AT 40 SECOND AND INTENSIFIED AT THE 230 SECONDS. WE ASSIGN THAT AS MAGNESIUM ION IN THE REACTION. A THIRD MAGNESIUM II DON'T KNOW. THE WATER IS VERY ESSENTIAL, BECAUSE IT TAKES THE PROTON -- WE PROPOSE IT TAKES THE PROTON AWAY. WHEN THE PROTON IS TAKEN WEIGH, IT CAN ATTACK THE ALPHA PHOSPHATE TO BREAK THIS BOND AND FORM A NEW BOND. THEN MARK THE GRADUATE STUDENT, HAS FURTHER STUDIED SHOWING THAT -- CONFIRMED THAT THE WATER ITSELF ISN'T A GENERAL BASE. WE CAN REPLACE THE WATER, WITH SOMETHING ELSE HERE, AND SO IT'S MAKE US TO PROPOSE THAT IT'S THE MAG DISEASIUMI ON -- MAGNESIUM ION. AND THE PATH HOW THE PROTON LEAVE VARIES DEPENDING ON THE ENVIRONMENT. IT'S READY TO LEAVE. DOESN'T SOLELY DEPEND ON THE WATER. AND -- AS THE REACTION GOES FORWARD, WHEN [INDISCERNIBLE] IS DEPROTNATED, IT HAS MORE CHARGE. THE TRANSITION STATE IS FAMOUSLY KNOWN TO HAVE CONCENTRATED CONNECTIVE CHARGE. WHAT WE SAW IS THAT THERE IS A THIRD MAGNESIUM ION. WHICH AT THE GROUND STATE IS ALTRACTING WITH ALPHABETA FOOT FATE. IN THE PRODUCT STATE, PLUS 2 CHARGE REPLACES THE PLUS 1 CHARGE THAT ALLOWS THE REACTION TO GO FORWARD. PEOPLE ASK WHETHER WE'RE CERTAIN THIS IS THERE, AND IF IT'S GENERAL FOR ALL DNA POLYMERASE. THE GOOD NEWS, THERE HAS BEEN AN EXTREME USING MAG IN A SEES, THOUGHIS THIS IS OBVIOUSLY THERE AND SHOWS UP EARLIER AND MORE OBVIOUS. AND SAME WILSON -- SAM WILSON IN NORTH CAROLINA HAS APPLIED THE SAME STRATEGY ON DIFFERENT DNA POLYMERASE AND IT ALSO SHOWED UP. SO IT COULD BE VERY GENERAL FOR ALL DNA POLYMERASE. ONLY FOR CAMPAIGN -- NOT ONLY FOR DNA POLYMERASE, ALSO FOR NUCLEAR LACE. MAGNESIUM ALIGNED THE SUBSTRATE. A DELAY, THE PROTENS CARRIED AWAY BY THE WATER. AND A REACTION PROGRESS, A THIRD MAGNESIUM COMING TO ENHANCE THE STABILIZED TRANSITION STATE AND PUSH THE REACTION TO THE PRODUCT STATE FORWARD. SO AS I SAID, THIS 3 [INDISCERNIBLE] IS GENERAL FOR NUCLEI ACE AS WELL. WE SAW IN TRANSIENT DURING THE PROCESS. LOOKING AT SUBSTRATE OF PRODUCT STATE, WE ONLY SAW TWO. FOR SOME, ACTUALLY SHOW THREE MEETAL IONS THROUGHOUT THE REACTION. COORDINATION IN THE ENVIRONMENT IS DIFFERENT. AND AT THE BEGINNING WE -- NOW WE THINK IT'S MORE GENERAL IN REPRESENTING THE TRUE TWO METAL ION WITH A TRANSIENT. THE STUDY OF GROUP ONE, AN RNA RIBOZYME, ABOUT 15 YEARS AGO, TWO GROUPS USING CHEMICAL METHOD, ANALYZING THE METAL BINDING PRO POPED THIS ARE THREE, A, B AND C, JUST AS HERE. BUT THEN THE CRYSTAL GRAPHERS SHOW THAT IN THE SUBSTRATE STATE AND PRODUCT STATE THEY'RE ONLY 2 METAL IONS. FOR A WHILE, PEOPLE WERE THINKING ONLY ONE CAN BE RIGHT. BUT NOW WE KNOW THAT BOTH ARE RIGHT. WHEN STRUCTURE BIOLOGISTS LOOK INTO SUBSTRATE OF PRODUCT STATE ONLY, THEY ONLY SEE THE TWO PERMANENT STABLE METAL ION, BUT THE CAN VOS, LOOKING AT THE WHOLE REACTION PROCESS, THEY CAPTURE THE TRANSIENT, WHICH WE CAPTURED. SO THIS IS ALL VERY WELL. AND THE LAST SLIDE I'LL RETURN TO REVERSE TRANSCRIPTASE, AND WE HAVE STUDIED, WHAT HAPPENED WITH THE TRANSCRIPT TASTE, WHICH IS -- TRANSCRIPT ACE, WHICH IS COMPLICATED. PEOPLE ARE THINKING THAT THE IN YOU ENZYMES CAN FIRE AT THE SAME TIME. HOW WONDERFUL, ONE END IS ADDING ONE END IS CHEWING. SO WE DIDN'T HAVE THE STRUCTURE FOR MANY YEARS. SO MARCIN, WHEN HE SAW THE STRUCTURE OF SIMILAR RNASE HE DID A MODELING EXERCISE. ALL PREVIOUS STRUCTURE, THE SUBSTRATE IS ENGAGED IN THE POLYMERASE ACTIVE CENTER. THE RNASE IS NOT CLOSE TO THE SUBSTRATE FOR CLEAVAGE. SO THE SIMPLE MODELING EXERCISE WAS JUST PUT THE CELLULAR RNASE IN ADDITION TO THE BACTERIA FORM, ALSO SIGNED THE HUMAN, AND FINDING THAT ACTIVE SITE IN PHOSPHATE BINDING SITE ARE VERY CONSERVED. THINGS CHANGE DOWN UNDER, NOT IMPORTANT. SO HE PUT THE TWO STRUCTURES TOGETHER AND SHOWED THAT THAT'S A CONSERVATION OF ALL RNASE ACTIVE SITE RECOGNIZED THE RNA STRAND AND YOU CAN SEE THE POUR BASE STRAND LAY ON TOP OF EACH OTHER VERY WELL, SO DOES THE DNA STRAND. SO HE TRIED TO BRING THIS INTO THE HIV RNASE STAGE. SO HERE IS THE HIVE REVERSE TRANSCRIPTASE, THE TWO CHAINS, LOCATED THERE. WE ROTATE THE MOLECULE BY 90 DEGREES, CAN BRING THE HUMAN RNASE HERE. AND THIS BIT OF HUMAN STRUCTURE IS NOT PRESENT IN HIV. AND, OF COURSE, SHOULD BE REMOVED. AND THE SUPER POSITION OF PROTEIN BRINGING THE RNA DNA STRAND INTO POSITION FOR CLEAVAGE. WHICH HAS NEVER BEEN SEEN BEFORE. AND SO BECAUSE THE PART OF THE PROTEIN IS DIFFERENT HERE, WE ANTICIPATE THIS WON'T FIT TO THE HIV R.T., SURELY IT DOESN'T. BUT IT'S MODELING EXERCISE, RIGHT? SO WE CAN JUST REMOVE THE PORTION THAT CLASHES BETWEEN THE SUBSTRATE, YOU CAN SEE CLASH WITH THE PROTEIN SURFACE. AND TRIM DOWN TO WHERE IT CAN FIT RNASE WELL. THEN BROUGHT IN WHAT HAS BEEN SHOWN OF THE COMPLEX WITH SUBSTRATE, WHEN SUBSTRATE IS IN THE DNA PHOSPHATE BINDING CENTER. SO THE DNA STRAND SUPPOSEDLY SHOULD BE MEETING EACH OTHER, AND THE PINK AND RED ARE THE RNA STRAND, THE TEMPLATE STRAND. THEY DON'T MEET. AND IT'S 17-OUNCE STRONG GAP IN BETWEEN. FULFILLING THE REQUIREMENT OF THE PHOSPHATE BINDING TO THE RNASE ACTIVE CENTER. AND THIS GAP CANNOT BE FILLED BY ANY AMOUNT OF BENDING AND WINDING, ET CETERA. SO THIS IS PHYSICAL EVIDENCE, RIGHT? STRUCTURE BIOLOGISTS, WE LOOK AT THE STRUCTURE. THIS CANNOT BE CHEAP. SOMETHING HAS TO GIVE. ONLY WAY WE CAN MAKE THINGS FORK IS THAT -- WORK IS THAT THE TWO ACTIVE CENTERS DO NOT FUNCTION, FIRE SIMULTANEOUSLY. AND SO LOOKING INTO THE LITERATURE. IF THERE IS ANY REPORT OF BIOCHEMICAL STUDIES, SHOWING THE ACTIVE DOES NOT FIRE SIMULTANEOUSLY. HE FIND PLENTY. THE TWO CATALYTIC CENTER WORK AT VERY DIFFERENT FREQUENCY. YOU COPY EVERY BASE OF RNA, BUT CLEARLY RNA HAPPENS ONCE IN A WHILE. THE TWO REACTION RATES ARE VERY DIFFERENT. RNA CLEAVAGE IS SLOW, OCCASION. DNA SYNTHESIS IS VERY FAST, CONTINUOUS. THERE IS A REPORT THAT RNA STAGE CLEAVAGE ONLY OCCUR WHEN THE DNA POLYMERASE IS NOT FUNCTIONAL. OTHER SUPPORTED EVIDENCE IS YOU CAN HAVE REVERSE TRANSCRIPTASE ONLY WITH ONE ACTIVE CENTER ACTIVE POLYMERASE. THE OTHER ONLY HAVE RNA H ACTIVE PUT INTO VARAN, THE VIRUS CAN STILL REPLICATE CONVERTING RNA GENOME TO DOUBLE STRANDED DNA. SO THEY DO NOT HAVE TO WORK SIMULTANEOUSLY. AND FINALLY, INHIBITION OF DNA POLYMERASE IN AN R.T.I. ENHANCES RNA STAGED CLEAVAGE. WE PROPOSE MAYBE THE ONLY WAY IT WORKS, THE TWO DOESN'T -- DO NOT WORK SIMULTANEOUSLY. AND FINALLY, I WILL SHOW YOU THE FINAL RESULT WE HAVE, RECENTLY. THAT WE HAVE A STRUCTURE THAT SHOWED HYBRID COMBATABLE FOR RNA CLEAVAGE, NOT FOR DNA SYNTHESIS. THE WORK WAS INITIATED BEA COLLABORATION AGAIN WITH OUR NIH COLLEAGUE, STEWART, HE HAD STUDIED RNASE FOR LONGER THAN I HAVE BEEN IN SCIENCE. HE PROVIDED PROTEIN INHIBITORS. VERY TALENTED SCIENTISTS IN ANY BROWN, GREW UP A THE CRYSTALS. AND IT'S A LONG DRAWN OUT PROCEDURE. LANCE SPENT THREE YEARS IMPROVING THE R.T. HIV R.T. WITH RNA DNA HYBRID IN THE PRESENCE OF AN INHIBITOR, ENHANCED RNASE CLEAVAGE. IN THE BEGINNING THE CRYSTAL DEFRACTURING TO 7. THERE WAS A TIME GO BY, WE CHANGE THE DNA CONSTRUCT, CHANGE THE PROTEIN, MUTATING DIFFERENT RESIDUES, AMP DN. ADN. IN 2009, IMPROVED THE DEFECTION TO 5 BUT IT'S NOT QUITE THERE STILL. ANOTHER YEAR-AND-A-HALF WORK, GOT A CRYSTAL THAT WAS WELL -- INHIBITOR AND DEFECTED TO X-RAY TO 3.3. AND THEN WE WERE ABLE TO SOLVE THE STRUCTURE WITH MOLECULAR REPLACEMENT. THE STRUCTURE THAT NICK SOLVED, SHOWING ON THE LEFT. THE NORMAL COMPLEX OF R.T. WAS SUBSTRATE IN THE CENTER ON THE RIGHT. AND ONLY POINTS 3 DIFFERENCES, FIRST, THIS IS THE CLOSE TO FORM AS WE KNEW, AS SHOWN BEFORE. THIS IS AN OPEN FORM. THIS HELIX SHOWING ORANGE, YOU CAN SEE THE POSITION IS VERY DIFFERENT. ALSO, THE SUBSTRATE LOOKS DIFFERENT. THIS IS MORE B FORMED, THIS IS MORE A FORMED. HYBRID CANNOT BE B FORMED. THE MOST INTERESTING THING, IF YOU FOCUS ON THE VERY END, THE CENTER THAT HIGHLIGHTS, THIS SUBSTRATES UP TOWARD THE RNA CENTER. THIS ONE IS SHOOTING DOWN. THIS IS ACTUALLY COMPATIBLE FOR CLEAVAGE. YOU'LL SEE IN A MINUTE. THIS IS A SUMMARY OF 25 STRUCTURES DEPOSITING THE DATABASE OF R.T. COMPLEX WITH DNA, DNA RNA. ALL OF THEM, BEFORE OURS, MORE OR LESS ENGAGED IN THE POLYMERASE CENTER. LOW AND HOLD, THE RNA IN ORANGE, DNA IN THE YELLOW. SUPER IMPOSABLE. WHAT'S ODD IS THE STRUCTURE PUBLISHED BY EDDY ARNOLD'S LAB CLAIMING TO BE RNA DNA HYBRID. WHEN WE POST ON TO THAT THE RNA STRAND SUPER IMPOSED PERFECTLY ON DNA WHICH IS SCIENTIFICALLY UNEXPLAINABLE. RNA COULDN'T BE TAKING THE B FORM AS DNA DOES. HOWEVER, OUR STRUCTURE CONTAINED RNA DNA HYBRID, THE RNA YOU CAN SEE SHOWING BROWN, VERY DIFFERENT. DNA IS MORE OR LESS SIMILAR, ALL DNA STRUCTURES. THIS JUST SHOWS THE BROWN COLOR DIFFERENT FROM ORANGE OR RED COLOR. AND THE NEXT THING I'LL SHOW YOU IS BY LOOKING AT THAT, MYSELF, WASN'T CONVINCED HOW DIFFERENT THIS STRUCTURE WE SAW IS FROM THE OTHER REPORTED STRUCTURE. BY MAKING THIS MOVIE IT'S NOW VERY OBVIOUS, THIS IS OUR STRUCTURE. THIS IS THE PREVIOUS STRUCTURE THAT'S COMPLEXED IN THE POLYMERASE ACTIVE CENTER. THE DUPLEX IS VERY MUCH, THE POLYMERASE ACTIVE CENTER SUBSTRATE. THIS IS RNASE ACTIVE CENTER STRUCTURE. YOU SEE THE UNWINDING OF THE DUPLEX AND BANDING. BIG CHANGE. AND THE CONSEQUENCE OF THIS BIG CONFIRMATIONAL CHANGE IS THAT NOW IN OUR STRUCTURE, THE RNA STRAND IS POSITIONED COMPATIBLE FOR RNASE. AND SO THIS STRUCTURE WE SOLVED AND RNA IN RED, DNA IS BLUE. WE SUPER INPOSED THE CELLULAR RNASE THAT WE KNOW IS ACTIVE, SUBSTRATE'S PERFECTLY POSITIONED INTO THIS COMPLEX. SO THE HUMAN RNA IS THE ORANGE RNA, DNA HYBRID COMPLEX WITH HUMAN RNASE IS SHOWING ORANGE. THE SUPER POSITION IS PERFECT, NO MORE GAP. IT CAN BE CONTIGUOUS. THE CAVEAT IS THAT OUR STRUCTURE IS NOT PERFECT. THE RAP RNA WE OBSERVED IS SLIGHTLY DIVIATED FROM ORANGE, PERFECTLY POSITIONED IN THE ACTIVE CENTER. 3 YEAR, 5 YEAR HARD WORK, GOT SOMETHING, NOT HOMEGROWN. WORK IS STILL WAITING FOR US TO GET THE SUBSTRATE PERFECTLY INTO THE ACTIVE CENTER. SO -- BUT IT SHOWS A VERY DIFFERENT CONFIRMATION OF NUCELAIC ACID. CONFIRMING OUR PROPOSAL FOR THE 2 PRO LIMNASE TO WORK, SUBSTRATE HAS TO TOGGLE AND IN BETWEEN THERE IS BIG CONFIRMATION CHANGE IN THE NUCELAIC ACID AS WELL AS PROTEIN. THE PROTEIN CHANGE IS HIGHLIGHTED HERE. THE MOST IMPORTANT ONES SHOWN, HIGHLIGHTED WITH FOUR RED ARROWS. THE BIG CHANGE IS ALWAYS CATCHING PEOPLE'S EYE. THE IMPORTANT SUBTLE CHANGE IS IMPORTANT FOR THE RNA DNA STRAND AND THE POSITIONING TO RNA EACH ACTIVE CENTER. SO THE SUBSTRATE BETWEEN THE 2 ACT I HAVE CENTERS IN THE REVERSE TRANSCRIPTATION, DEPENDING ON THE CHANGE IN THE MIDDLE WHICH IS NEITHER THE POLYMERASE NOR THE RNASE ACTIVE CENTER. WHAT'S INTERESTING, WITH OUR STRUCTURE, NOW WE CAN EXPLAIN WHY VARIOUS DRUG RESISTANT MUTATIONS OCCUR IN THE MIDDLE OF THE PROTEIN. RATHER THAN IN THE BINDING SITE OF THE DRUG. THE REASON IS BY MUTATING THE MIDDLE PORTION WHICH CRITICAL CONFIRMATIONAL CHANGE GOING FROM ONE CENTER TO ANOTHER, IT'S BECOME FAVORED SUBSTRATE IN THE POLYMERASE CENTER. BY DWELLING IN THE POLYMERASE ACTIVE CENTER, A CHANCE TO GET IT RIGHT, TO REMOVE OR RETRY THE SYNTHESIS. SO THIS IS THE COMBINATION OF RNA DNA SUBSTRATE CHANGE WEST PROTEIN STRUCTURE CHANGE. IT EXPANDED TO ALLOW THE SUBSTRATE TO ALLOW A DIFFERENT CONFIRMATION ENABLED RNASE CLEAVAGE. OUR WORK IS STILL CUT OUT FOR US, AND WE ARE CURRENTLY TRYING TO, WORKING WITH STEWART, SCREENING MANY, MANY HIV DRUGS THAT BINDING OUTSIDE OF POLYMERASE ACTIVE CENTER, IS PREVENTING THE CONFIRMATIONAL CHANGE OR RNASE ACTIVITY. AND WORKING VERY HARD, TRYING TO GET A HOME RUN. THE REAL SUBSTRATE IN THE REAL ACTIVE CENTER. SO THANK YOU VERY MUCH. FOR YOUR ATTENTION. AND I'VE ACKNOWLEDGED PEOPLE AND MAY HAVE MISSED A FEW ALONG THE WAY. THE TEAM IS THE PAST MEMBER IS EXCEEDING THE CURRENT MEMBER, HOW LIFE GOES. AND I THANK ALL MY COLLEAGUES AND NIH FOR THE VERY STRONG SUPPORT AND MAKING, WORKING HERE FUN. THANK YOU. [APPLAUSE] >> I'M SURE WEI WOULD BE HAPPY TO ANSWER QUESTIONS IF THERE ARE ANY. >> THE STRUCTURE WITH THE DNA HYBRID, IN THE MOVIE I SAW AT THE END, THERE IS A GAP IN THE RNA. HAS YOUR PROTEIN CLEAVED RNA ALREADY? >> NO. SO I SKIPPED THAT. SO FOR OUR CRYSTALLIZATION SCREENING WE TRIED A DIFFERENT LENS, SO WE PURPOSELY PUT A NICK IN RNA STRAND TO CHANGE THE LENS MORE EASILY. WE DON'T HAVE TO CHANGE THE FULL RNA. >> CAN YOU ACTIVATE THAT NICK TO THE FACT THAT THE RNA IS NOT BEING ABLE TO GET TO THE ACTIVE SITE? >> WE HAVE ACTUALLY TWO OTHER CRYSTAL FORMS. WHICH WERE DETERMINED AT THE LOWER RESOLUTION. ALONG THE WAY I SHOWED YOU THE SLIDE. THE OTHER TWO CRYSTAL FORMS, THE RNA DIDN'T HAVE A NICK AND PACKING DIFFERENT SPACE GROUP. THE FEATURE OF RNA DNA HYBRID ARE SIMILAR. >> I SEE. >> SO IT'S NOT DUE TO THE NICK. AND IT'S CRYSTAL PACKING. STILL TRYING TO FIGURE OUT. >> THE OTHER QUESTION I HAD, FOR THESE R.T., I CAME TO THE WORK THAT [INDISCERNIBLE] HAS DONE SHOWING THAT FAR FROM THE ACTIVE SITE, THEY CAN SENSE CHANGES IN THE DNA STRUCTURE. DO YOU HAVE A FEELING WHEN WHETHER THESE ARE DIFFERENT CONSIDERING THEY'RE MORE TOLLANT ACT OF SITES. DO YOU HAVE ANY THOUGHTS ON THAT. >> MISMATCH IN THE DNA DUPLEX EFFECT HOW INFERENCE HOW THE ACTIVE SITE IS FORMED. IT DISTORTING IT. YES, THAT'S TRUE. BUT IF THE DNA DUPLEX IS PERFECT, THE ACTIVE SITE DOESN'T MATTER, IT'S INCORPORATING A, T., C, G, IT'S IDENTICAL. THE REACTION IS THE SAME. WE PARTICULARLY ADDRESSED THE CRYSTAL COMPACT, WE'RE GENERATING TWO DIFFERENT CRYSTAL LATTES. AN WE MONITOR THE REACTION. IT'S THE SAME. THE TIME OF THE BOUND FORMATION WAS THE SAME. SO IT'S VERY REASSURING. PARTICULARLY CRYSTAL FORMED. WE PRESERVE THE ACTIVE CENTER WITHOUT DISTURBING IT BY LATTICE COMPACT SO THE REACTION CAN OCCUR INDEPENDENT. >> HI. SO I JUST HAD SORT OF A TECHNICAL QUESTION. CURIESTTY QUESTION. I WAS AMAZED BY THE TIME LAPSE STRUCTURE THAT YOU SHOWED. I WAS WONDERING IF WHEN YOU DO THIS KIND OF SOAKING PROCEDURE, DO YOU END UP WITH HETEROGENEOUS STRUCTURES? IS THAT A PROBLEM. IS THIS A KIND OF APPROACH YOU CAN TAKE GENERALLY? OR IN THE SPECIFIC CASE? >> VERY, VERY GOOD QUESTION. FOR LARGE CONFIRMATIONAL CHANGE THEY'RE BOUND TO BE HETEROGENEITY. LUCKILY, IN OUR CASE, WE DON'T HAVE LARGE CONFIRMATIONAL CHANGE AT ALL. THE ONLY THING CHANGE, IN ACTIVE CENTER, IS ONLY THE BOUND GETTING MADE AND ONE BOND GET BROKEN. AND SO THE REACTION THERE ARE HUNDREDS, THOUSANDS OH MOLECULES, CRYSTAL. THE REACTION FOR EVERY MOLECULAR MAY TAKE DIFFERENT TIME POINT. BECAUSE SLOWLY, FASTER. BUT IN THE REACTION CENTER WE ONLY SEE TWO DIFFERENT STATE. BEFORE REACTION AND AFTER REACTION. SO THE HETEROGENEITY BETWEEN THE PERCENTAGE OF INCREASING OF THE PRODUCT AND DECEDESING OF PRODUCT, OF SUBSTRATE. IT'S VERY EASY TO MODEL. WE HAVE MORE DATA THAN NEEDED TO CHEER LEE DEFINE THOSE TWO STATES. WE ARE NOT KNOWING EXACTLY THE TRANSITION STATE, WHICH IS TRANSIENT. WE DIDN'T TRY TO MONITOR THE INTERMEDIATE BETWEEN SUBSTRATE TO PRODUCT STATE. IT SHOULD BE GENERALLY APPLICABLE. WE'RE WORKING ON R.T. RIGHT NOW ON THE POLYMERASE AND RNASE. >> I WAS REALLY INTRIGUED BY THE TRANSIENT MAGNESIUM IN THE HYDROLYSIS REACTION. DO YOU THINK THAT COULD BE HAPPENING IN KINASES AND OTHER HYDRO LIESING PROTEINS AS WELL? >> YES. SO IT COULD BE BUT WE -- I THINK REALLY REQUIRE EVIDENCE, AND SO WE ARE CURRENTLY WORKING ON NU CLASS. WE HAVEN'T LOOKED AT -- IT'S POSSIBLE. ONE OF THE [INDISCERNIBLE], THE ACTIVE CENTER IS HO MODELGOUS TO RNASE SO WE'RE LOOKING AT RIBONUCLEAR RNASE AND HOPEFULLY WE CAN ADDRESS THE QUESTION IS REAL EVIDENCE. >> I WAS DOING SOME KINASE COMPUTATIONAL STRUCTURAL BIOLOGY. WE DID A SERIES OF COMPUTATIONS THAT SHOWED THAT THE MAGNESIUMS ARE VERY, VERY LOW FREE ENERGY. WE COULDN'T FIGURE OUT HOW THE ADP COULD LEAVE THE ACTIVE SITE, BUT IF THERE IS A TRANSIENT MAGNESIUM THAT COMES IN, THAT WOULD EXPLAIN HOW THE MAGNESIUM ADP CAN GET OUT AFTER THE CATALYTIC REACTION. >> WE'D VERY MUCH LIKE TO SEE THAT HAPPEN. THE REASON IS IT DOESN'T MATTER, THE TRANSITION STATE IS SAME. HIGHLY CHARGED CONNECTIVE STATE. SO A TRANSIENT METAL ION WOULD HELP THE PRODUCT TO RELEASE. >> THANK YOU. >> I THINK I WOULD -- DETAILS THAT SHOW THIS VERY NICE CRYSTAL STRUCTURE, HOW PRECISE [INDISCERNIBLE] MAKES IT WORK IN THE COMBINATION [INDISCERNIBLE]. THE QUESTION IS, IN THE REAL WORLD, IN THE CELLS, POLYMERASE WITH MANY OTHER VIRAL PROTEINS, HOW THE DETAILS MAKE US TO THINK ABOUT IN THE CELLS, EFFECTED BY OTHER PROTEINS, ESTABLISH DIGESTIONS. COME FROM A BROADER FUNCTIONAL STUDY. >> RIGHT. SO IT'S NICE TO HAVE VERY CLOSE UP VIEW TO UNDERSTAND HOW THINGS WORK. AND THEN IN TERMS OF WHAT WE CAN USE OUR KNOWLEDGE FOR, IN TERMS OF TREATING PATIENTS OR ANTI-HIV THERAPY, IN A WAY, ACTUALLY CERTAIN MEETAL ION IS A GOOD HINT THAT NUCLEOTIDE ANALOG INHIBITORS, MAYBE CAN BE SYNTHESIZED WITH A METAL MIMMICK, SO THAT CAN BIND THE INHIBITOR R.T. MORE EFFECTIVELY. HAVE A MORE BINDING SITE. I WERE TERMS OF CELLULAR FUNCTION, UNDERSTANDING THAT HELPS US TO UNDERSTAND WHY WE NEED MAGNESIUM. >> WELL, BECAUSE [INDISCERNIBLE] TALKING ABOUT A AND MANY OTHER THINGS. ALSO TALKING ABOUT MANY OTHER PROTEINS. THE FUNCTION OF -- >> YES. OKAY. SO OTHER PROTEIN INTERACTION CAN CHANGE THE TIMING, THE RECRUITMENT, WHEN THINGS HAPPEN, CAN BE INHIBITED, THE WHOLE PROCESS OF REVERSE TRANSCRIPTION. BUT WHEN IT GETS DOWN TO THE BUSINESS, DOING THE DNA SYNTHESIS, AND THE CONVERTING RNA GENOME TO DNA, THAT'S WHAT HAPPENS. OTHER CONTROLS WILL CONTROL THE PROCESS. THIS IS JUST THE WORK THAT WE FOCUS ON, NOW, IF YOU KNOW ANY PROTEIN THAT REGULATE REVERSE TRANSCRIPTION, PLEASE LET US KNOW. >> ALL RIGHT. LET'S THAT I WEI YANG AGAIN FOR A FANTASTIC LECTURE.