Download SF Genetics Lecture_Central Dogma McConnell_4.2 BY2208

The Central Dogma
Protein structure and
synthesis
How does a mRNA “make” a protein?
What is the mechanism of protein synthesis?
David McConnell
Smurfit Institute of
Genetics
Clue #1
The Discovery that Proteins are made on
ribosomes
•! By Paul Zamecnik (early 1950s)
•! He asked where in the cell are proteins
synthesized?
•! Injected rats with radioactive amino acids
•! A short time after injection (when the amino acids
should be incorporated into newly-synthesized
proteins) he killed the rats, harvested their livers,
ground them up and separated the cell components
into “subcellular fractions” by centrifugation
Results
•! Radioactivity was found in small ribonucleoprotein
particles visible by electron microscopy.
•! These were later characterized and called
“ribosomes” (since they had RNA as a major
component - this is ribosomal RNA - rRNA)
From Lehninger “Principles of Biochemistry” p 1021"
Ribosomes
Protein factories
Themselves made of many different proteins
and a few RNA molecules
Ribosomal RNA
Ribosomal proteins
Macromolecular machines
Bacterial Ribosomes
S value
(Svedberg value)
“Sedimentation coefficient”
Measure of sedimentation of a particle
at 20O in water
Larger particles have higher S values
Affected by shape and density as well
as size
S values are not additive
Ribosome structure 2
rRNA"
rRNA secondary structure
Proteins"
Sub-units"
P
r
o
k
a
r
y
o
t
e
s
Ribosomes"
E
u
k
a
r
y
o
t
e
s
Clue #2
Ribosomes
A ribosome is a general purpose
protein factory
No specificity
Specificity provided by
mRNA and tRNA
The Discovery that amino acids are
“Activated”
•! By Hoagland and Zamecnik
•! They incubated amino acids with the cytosolic fraction
of liver cells, and with ATP
•! They found the amino acids became “activated” during
the incubation
•! Activation consists of attaching the amino acids to a
heat-stable soluble RNA (which we now know is tRNA)
•! Activated amino acids are called aminoacyl- tRNAs
•! The enzymes that do the activation are called
aminoacyl-tRNA synthetases
Clue #3
Crick’s Adaptor Hypothesis
Crick’s Adaptor Hypothesis
•! Must have one adaptor
per amino acid"
•! Therefore there must be
a family of adaptors:"
•! Francis Crick reasoned that a small nucleic acid could serve as
an adaptor between RNA and protein synthesis if it could bind
both RNA and an amino acid
•! These are the tRNAs"
•! Each tRNA can
recognize specific
sequences in the RNA
transcript"
•! His idea was that one end of the adaptor would bind a specific
amino acid and the other would bind to a specific sequence in
the RNA that coded for that amino acid
•! Each is “charged” with
the amino acid that is
specified by that sequence"
From Lehninger “Principles of Biochemistry” p 1021"
Review of tRNA Structure
•! tRNAs are the “adaptors” in protein synthesis
•! There are many different tRNAs, each has a distinct
sequence
•! However, all tRNA have several conserved features
1) small (73-93 nucleotides long)"
2) they have a conserved secondary structure - 4
stems and 4 loops with important functions"
3) they contain many unusual bases"
Inosine (I), pseudouridine (!), dihydrouridine (D),
ribothymidine (T), and methylated bases (mG, mI)"
•! An aminoacyl-tRNA synthetase attaches an
amino acid to its tRNA
Figure 9-8
tRNA charging reaction
tRNAX
+ ATP +
amino acidX"
+ "
aminoacyl tRNA synthetase"
aminoacyl tRNAX"
+"
AMP"
+"
PPi"
tRNA charging reaction
•! The structure of transfer RNA
tRNA charging reaction
31 - 61 tRNAs
20 amino acids
20 aminoacyl tRNA synthetases
tRNA has modified bases
tRNA has modified bases
Modifications occur
post-transcriptionally
Example
Catalysed by a variety of
enzymes
Guanine to Inosine
tRNA tertiary structure: banana or L-shape
Noncanonical base pairs stabilize the 3o structure"
Crick’s Adaptor Hypothesis
Adaptor is tRNA"
Each codon is read
by at least one tRNA"
Wobble allows some
tRNAs to recognise
more than one
codon "
From Lehninger “Principles of Biochemistry” p 1021"
Wobble allows one tRNA to recognize
more than one codon
tRNA anticodon
loop
3’ Anticodon 5’
Wobble base
5’ Codon 3’
5’ mRNA 3’
Non-canonical base pairs - Wobble - occur in RNA
3 seryl tRNAs recognise 6 Serine codons
3#"
5#"
5#"
3#"
AGG"
Protein synthesis reactions
A specific mRNA
A ribosome
Pool of charged tRNAs
GTP
Initiation Factors (IF), Elongation Factors (EF)
Release Factors (TR)
•! Key sites of interaction in the ribosome
Figure 9-13
•! Key sites of interaction in the ribosome
•! Key sites of interaction in the ribosome
Nobel Prize "
Chemistry 2009"
Nobel Prize "
Medicine 2009"
Venkatraman Ramakrishnan
Thomas A. Steitz
Ada E. Yonath
Elizabeth H. Blackburn, Carol W. Greider
and Jack W. Szostak
Atomic structure
of the
Ribosome
for the discovery of
"how chromosomes are protected
by telomeres
and the enzyme telomerase"
Protein synthesis reaction
RNA is decoded in the 5’ to 3’ direction
Protein is synthesised
in the
N terminal to C terminal direction
•! Key sites of interaction in the ribosome
Figure 9-13b
•! Translation initiation in prokaryotes
Initiation "
of "
protein synthesis"
AUG codon"
Formylmethionyl tRNA"
mRNA"
Most proteins "
start with MET"
Most genes start with"
AUG codon"
•! Translation initiation in prokaryotes
•! Steps in translation elongation
Assembly "
of"
initiation "
complex"
(bacteria)"
•! Termination of translation
•! Key sites of interaction in the ribosome
Termination "
of"
Translation"
STOP CODONS"
UAG - amber"
UAA - ochre"
UGA - opal"
mRNA"
Dintzis Experiment
Globin synthesis
Protein is synthesised
in the
N terminal to C terminal direction
Pulses of radioactive amino acids
Purify the ribosomes with attached mRNA and
nascent protein
Proof from the Dintzis Experiment
Purify soluble globin
Locate the radioactivity
Polysomes 1
EM
Dintzis Experiment
Polysomes EM
“Double Helix”
Brian King
Unveiled by
James Watson
April 2003
50th birthday of
The Double
Helix
Gift of Dr.
Beate Schuler
Nobel prizewinners in Chemistry
related to Genetics and Molecular Biology
Nobel prizes related to genetics"
Peace Prize"
Norman Borlaug"
1970"
Green Revolution"
2009 2008 2006 2004 1993 1989 1980 1962 1958 -
Ramakrishnan, Steitz, Yonath
Shimomura, Chalfie, Tsien
Kornberg
Ciechanover, Hershko, Rose
Mullis, Smith
Altman, Cech
Berg, Gilbert, Sanger
Perutz, Kendrew
Sanger
Genetics
Genetics
Nobel prizewinners in Medicine 1933-71
related to Genetics and Molecular Biology
1971 1969 1968 1965 1962 1960 1959 1958 1946 1933 -
Sutherland
Delbruck, Hershey, Luria
Holley, Khorana, Nirenberg
Jacob, Lwoff, Monod
Crick, Watson, Wilkins
Macfarlane Burnet, Medawar
Ochoa, Kornberg
Beadle, Tatum, Lederberg
Muller
Morgan
Nobel prizewinners in Medicine 1972-86
related to Genetics and Molecular Biology
1986 1985 1984 1983 1980 1978 1976 1975 1974 1972 -
Cohen, Levi-Montalcinii
Brown, Goldstein
Jerne, Köhler, Milstein
McClintock
Benacerraf, Dausset, Snell
Arber, Nathans, Smith
Blumberg, Gajdusek
Baltimore, Dulbecco, Temin
Claude, de Duve, Palade
Edelman, Porter
Nobel prizewinners in Medicine 1987-2009
related to Genetics and Molecular Biology
2009 2008 2007 2006 2004 2002 2001 1997 1995 1993 1989 1987 -
Blackburn, Greider, Szostak
zur Hausen, Barre-Sinoussi, Montagnier
Capecchi, Evans, Smithies
Fire, Mello
Axel, Buck
Brenner, Horvitz, Sulston
Hartwell, Hunt, Nurse
Prusiner
Lewis, Nusslein-Volhard, Wieschaus
Roberts, Sharp
Bishop, Varmus
Tonegawa
Thank you for listening and good luck
David McConnell
[email protected]