Transcript Document

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