Project Grant F32AI194768

EXPLORING LEADERLESS MRNA TRANSLATION, A POTENTIAL ACHILLES HEEL OF MYCOBACTERIA - PROJECT SUMMARY/ABSTRACT TUBERCULOSIS (TB) REMAINS A SIGNIFICANT GLOBAL HEALTH THREAT, CAUSING APPROXIMATELY 1.5 MILLION DEATHS ANNUALLY, WITH TREATMENT COMPLICATED BY THE RISE OF DRUG-RESISTANCE MYCOBACTERIUM TUBERCULOSIS (MTB) STRAINS. A COMMON TARGET FOR ANTIBIOTICS IS THE BACTERIAL RIBOSOME, AND MYCOBACTERIAL RIBOSOMES HAVE UNIQUE FEATURES AND FUNCTIONS, WHICH MAY BE EXPLOITED TO DEVELOPMENT MORE EFFECTIVE TREATMENTS. ONE SUCH EXAMPLE IS THE PREVALENCE OF SO- CALLED LEADERLESS MRNAS, (LMRNA) WHICH LACK THE 5' UNTRANSLATED REGION AND SHINE-DALGARNO SEQUENCE USED DURING CANONICAL PROTEIN TRANSLATION INITIATION. THIS PROJECT SEEKS TO DEVELOP AN UNDERSTANDING OF THE MECHANISM OF LMRNA TRANSLATION IN MYCOBACTERIA AND UNDERSTAND HOW THIS FORM OF TRANSLATION IS REGULATED. I HYPOTHESIZE THAT THERE ARE UNIQUE STRUCTURES IN THE MYCOBACTERIAL RIBOSOME THAT ALLOW FOR EFFICIENT TRANSLATION OF LMRNAS AND THAT LMRNAS ARE OF PARTICULAR RELEVANCE DURING STRESS OR INFECTION CONDITIONS. AIM 1 WILL USE REVERSE GENETICS TO SCREEN CANDIDATE REGULATORS OF LMRNA TRANSLATION IN MYCOBACTERIA USING A PAIR OF ORTHOGONAL REPORTERS FOR LEADERLESS TRANSLATION. REGULATORS VALIDATED BY BOTH REPORTERS WILL BE FURTHER INVESTIGATED BY RIBOSOME PROFILING. AIM 2 WILL USE FORWARD GENETICS IN THE FORM OF A CRISPRI SCREEN TO IDENTIFY UNKNOWN REGULATORS OF LMRNA TRANSLATION IN MYCOBACTERIA USING A LEADERLESS SACB REPORTER. HITS FROM THE SCREEN WILL BE VALIDATED USING AN ORTHOGONAL FLUORESCENT LEADERLESS TRANSLATION REPORTER, AND CONFIRMED HITS WILL BE FURTHER ANALYZED USING RIBOSOME PROFILING. AIM 3 WILL LEVERAGE CRYOEM TO IDENTIFY STRUCTURAL COMPONENTS OF THE MYCOBACTERIAL RIBOSOME THAT CONTRIBUTE TO EFFECTIVE LEADERLESS TRANSLATION, FIRST BY ISOLATING AND COMPARING STRUCTURES OF THE RIBOSOME ON CANONICAL AND LEADERLESS MRNA TRANSCRIPTS AND LATER BY SOLVING STRUCTURES OF RIBOSOMES LACKING PROMISING VALIDATED HITS FROM AIMS 1 AND 2. THIS WORK WILL ALLOW FOR THE IDENTIFICATION OF NOVEL THERAPEUTIC TARGETS ON THE MYCOBACTERIAL RIBOSOME, SUPPORTING THE BROAD NIH MISSION TO REDUCE THE BURDEN OF INFECTIOUS DISEASES. THIS PROJECT COMBINES MY RESEARCH INTERESTS IN INFECTIOUS DISEASE, PROTEIN TRANSLATION, AND BACTERIAL GENETICS WHILE INCREASING MY FAMILIARITY WITH STRUCTURAL BIOLOGY. THIS AWARD WILL SUPPORT MY LONG-TERM CAREER DEVELOPMENT INTO AN INDEPENDENT ACADEMIC RESEARCHER, AND I WILL SUPPLEMENT IT WITH CONTINUED MENTORING AND TEACHING, AS WELL AS TAKING ADVANTAGE OF THE CAREER AND PROFESSIONAL DEVELOPMENT PROGRAMS AVAILABLE AT UCSF.