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Gene Expression Central Dogma & Mechansim Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Archibald Garrod (Scottish physician, 1908) • Some inherited diseases due to absence of enzyme • inborn errors of metabolism • Alcaptonuria – – – – urine gets dark upon exposure to air lack enzyme that oxidizes homogentisic acid excess excreted into urine & oxidizes in air breakdown product of phenylalanine & tyrosine Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E George Beadle & Edward Tatum (Caltech, 1940s) • used Neurospora – – – – – – tropical bread mold grows in a simple medium makes all its required metabolites mutated mold spores with irradiation test on minimal medium and supplemented media uncovered metabolic/enzymatic deficiency • Formulated one gene - one enzyme hypothesis – later modified to one gene - one polypeptide – alternative splicing: related polypeptides Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.1 Vernon Ingram (Cambridge, 1956) • cleaved normal & sickle cell hemoglobin with protease • trypsin (~30 fragments) • separated by paper chromatography • one fragment from sickle cell migrated differently • valine substituted for glutamic acid Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.2 messenger RNA • • • • • • • • 1961 Jacob & Monod (Pasteur Institute, Paris) mRNA complementary to 1 of 2 DNA strands mRNA code read during translation Read by ribosomes in cytoplasm (large & small subunits) Ribosome subunits join when synthesis starts Ribosomes made of protein & ribosomal RNA Many mRNAs per gene ; many proteins per mRNA Amplifies output from gene Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E rRNAs • Adapted to recognize & bind other molecules • Provide structural support • Catalyze chemical reactions of translation Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.3 Transfer RNAs (tRNAs) • tRNAs & rRNAs have long half-life in cell (days) • rRNAs, tRNAs have complex secondary & tertiary structures • RNAs fold into complex 3D shapes • RNAs carry out a diverse array of functions • double stranded & double helical stems & • single-stranded regions (loops) • often contain nonstandard mismatched base pairs • often contain modified nitrogenous bases Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Transcription: The Basics • Begins with association of RNA polymerase & DNA template – RNA polymerases bind to promoters – they cannot recognize promoters on their own – but require help of other proteins (transcription factors) – TFs particularly important in initiation of eukaryotic gene transcription – Promoter specifies which strand Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Transcription: The Basics • Polymerase lays down complementary RNA – – – – – – – – – – new strand 5' —> 3' direction template DNA in 3' —> 5' direction unwinds DNA temporarily as it travels along essentially irreversible exergonic pyrophosphate hydrolysis must have proper match to template DNA helix closes behind polymerase only few (~8) RNA bases attached at a time Bacteria incorporate 50 - 100 nucleotides/sec; genes in cell transcribed at same time by numerous polymerases Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.4a-c Transcription: The Basics • • • • • • • • • • RNA pol enzyme is processive must bind tightly, but not too tightly Movement like cytoskeletal motor kinesin fluorescent bead covalently linked to one end monitor bead movement Allows determination of transcription rate trap bead by laser beam & measure force force >2X that of myosin interacting with actin Energy comes from NTP precursor hydrolysis RNA pol may pause at some sites (Proof reading) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.5 Transcription in Prokaryotes • E. coli has single RNA polymerase • 4 subunits associate tightly forming core enzyme of E. coli RNA Polymerase – Can purify core enzyme – It starts synthesis at random sites • Purify accessory sigma (s) factor & add – Transcription starts at right places – Transcription begins, s leaves & core continues Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.6 Transcription in Prokaryotes • X-ray crystallography analysis of core – reveals shape: crab claw with a broad internal channel – A pair of jaws grips DNA as it enters the enzyme – Enzyme “melts” the 2 DNA strands at start site – This makes the template strand accessible – Polymerase can make several unsuccessful attempts – After ~10 nucleotides, release of s factor – Elongation complex that can move processively Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Transcription in Prokaryotes • Archaea – also possess a single RNA polymerase – very different subunit composition – more similar to eukaryotic RNA polymerase Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Transcription in Prokaryotes • Bacterial promoters – located in region of DNA strand just preceding initiation – transcription initiates at +1 – preceding base is –1 – DNA preceding initiation site “upstream” – DNA succeeding initiation site “downstream” Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Transcription in Prokaryotes • Promoter elements: consensus – -35 region TTGGACA & -10 region TATAAT – -35 region recognized by s factor – different s factors recognize different –35 sequences • The s70 factor is housekeeping s factor • it initiates transcription of most genes – Alternative s factors • small number of specific genes • specific response • one for the heat shock genes Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.7 Transcription in Prokaryotes • -10 region TATAAT – Pribnow box – Determines initiation site and strand • Most mutations reduce transcription rate – certain mutations can greatly increase transcription – each promoter tuned to level of expression Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Transcription in Prokaryotes • Termination of transcription – – – – – specific sequence signals termination stem-looe structure Sometimes rho factor protein is required rho dependent terminators rho independent terminators Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E