Protein Synthesis
E. coli Ribosome
-70S particle, MW ~2.5 x 106
-dissociable into small (30S) and large (50S) subunits
-30S contains 16S RNA, 21 polypeptides
-50S contains 5S, 23S RNA + 31 polypeptides
“Although the ribosome has been crystallized…it is such
a complex entity that it will be many years before its
structure is known in molecular detail”
- Voet and Voet, Biochemistry 1995
Ribosome X-ray Structure
Science (2000) 289:920-30
Puromycin Inhibition of Protein Synthesis
H3 C
N
N
N
HO
CH3
H
N
N
N
N
tRNA-O3 PO
O
O
CH3 O
HO
+
NH3
NH
H
N
+
NH3
O
OH
O
Puromycin
“…is known to interfere with protein formation by interfering
with the function of RNA in the cells involved. In goldfish studied
by Bernard W. Agranoff at the University of Michigan long term
memory was obliterated when the fish were given minute injections
of puromycin. Since short-term memory is not much affected, it is
concluded that the antibiotic interferes with the process by which
memory becomes fixed in the brain” - Merck Index 1968
OH
O
Tyrosyl-tRNA
N
N
Science (2000) 289:920-30
Science (2000) 289:920-30
Chemistry of Peptidyl Transfer
B
B
B
Science (2000) 289:947-50
Split Genes and RNA Splicing
Nobel Prize - Medicine or Physiology - 1993
studies on genetic structure of adenovirus 2
P.A. Sharp (Biology, MIT)
Proc. Natl. Acad. Sci. U.S.A. (1977) 74, 3171-5
Split Gene Structure
-eukaryotes from yeast to humans
-90% of human genes
-intron length highly variable
-exon length ~200 nt
-Dystrophin
Gene Structure Analysis by EM
mRNA
?
template DNA
Discovery of Split Genes (1977)
chicken ovalbumin
7700 bp
mRNA
I
III
1
5’
DNA
IV VI
3 45
6
2
VII
1872 nucleotides
< 20% coding
II
7
V
3’
Voet and Voet Biochemistry
Pre-mRNA Splicing
-splicing is nuclear (HeLa nuclear extracts)
-requires Mg2+
-requires ATP
-(is co-transcriptional)
Analysis of in vitro Splicing of 32P-Labeled pre-mRNA
IVS-E2
IVS
E1-IVS-E2
E1-E2
E1
Pre-mRNA Splicing
Chemistry of Splicing
1st step
2nd step
5’ exon
O
G
O
O
5’ exon
O
-O
O
O P
O
-O
OH
OH
O
O
OH
G
O
G
O
G
O
OH
P
O
G
O
OH
O
O
OH
O
O
O
OH
3’ exon
A
5’ exon
O
G
O
5’ exon
O
G
O
O
O
O
A
OH
-
O O
O P
OH
OH
OH
O
G
O
-
O O
O P
OH
O
G
O
O
G
O
O
O
OH
OH
3’ exon
Splicing Time Course
IVS-E2
IVS
E1-IVS-E2
Complex Formation in HeLa Extract
C
B
A
H
Discovery of the Spliceosome
-60S particle required for pre-mRNA splicing
-spliceosome contains ribonucleoprotein particles
(snRNPs - small nuclear)
-U1, U2, U4/U6 U5
-each snRNP contains respective snRNA (U1, U2,
U4/U6 U5) + associated proteins
Cell (1985) 42, 345-53
snRNP Composition
Protein Components of the Spliceosome
-~10-220 kD
-structural roles, functional roles
-conserved (core), unique
-non-snRNP
“Comprehensive proteomic analysis of the human spliceosome.”
Nature 419, 182-185 (2002).
snRNP Core Proteins
B,D1,D2,D3,E,F,G
Cell (1999) 96, 375-87
The spliceosome cycle
5' SS
bp
3' SS
Py
5'-
exon 1
AG exon 2 -3'
A
GU
Pre-mRNA
CC
5'-
U1
GU
A
-3'
AG
U1
A
U1
ATP
U2
-5'
UG
U2
A
-3'
AG
U4/U6
U4/U6
U5
U4
A
-5'
U1
U4/U6
U6
U1
AG
B1
U2
U5
U6
U5
U2
A
-3'
U4
U5
A
-5'
U4
AG
B2
U4
U1
U6
U5
I
C1
U2
A
C2
U4
ATP
U6
U5
U2
A
5'- exon 1
exon 2 -3'
mRNA
-3'
U2
A
ATP
-5'
U4
U1
U6
-5'
U1
U1
U6
U2
A
U5
-3'
U5
-3'
Splicing Directed by Conserved Intron Sequences
5’ splice site
3’ splice site
branch region
poly-pyrimidine tract
.
H
eL
 a
U
2
 AF
U
2A
F
+
U
2A
F
Early Steps in Spliceosome Assembly
Recognition of the Pyrimidine Tract
U2AF required for A complex
U2 Auxiliary Factor
-heterodimer, 65 kD, 35 kD subunits
-U2AF65 required splicing factor
-U2AF35, 3’ splice site
U2AF65 Domain Structure
MSDFDEFERQLNENKQERDKENRHRKRSHSRSRSRDRKRRSRSRDRRNRDQ
RSASRDRRRRSK-
U2AF65
Branch Region
Pyr tract
Bridged
Commitment
Complex
Bridged
Commitment Complex
E Complex
UG
U1snRNP
SR
BBP
A
U2AF65
Py
SR
U2AF35
AG
.
EBC
Selection of 5’ Splice Site and Branch
5' splice site
5' exon GUAUGU
••• ••
CAUUCA
U1
BBP
UACUAAC
5'
3' splice site
3' exon
U2
A
UACUAC
•••• ••
AUGAUG
34
3' exon
5'
RNA Rearrangements in the Spliceosome
-extensive U4/U6 interaction is replaced
with a U2/U6 structure
-U1 displaced at 5’ splice site by U6
RNA Rearrangment at 5’ Splice Site
U6
5' splice site
5' exon GUAUGU
••• ••
CAUUCA
3
U1
47
GACACA
•••
5' exon GUAUGU
5'
5'
Unusual Classes of Introns
“AT-AC”
minor spliceosome