E P A E P A

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

“…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

O

N

NN

N

CH3N

H3C

HO

NH OH

CH3O

O

NH3+

Tyrosyl-tRNA

ON

NN

N

HN

H

tRNA-O3PO

O OH

HO

O

NH3+

Puromycin Inhibition of Protein Synthesis

Science (2000) 289:920-30

Science (2000) 289:920-30

Chemistry of Peptidyl Transfer

Science (2000) 289:947-50

BB

B

Split Genes and RNA Splicing

Nobel Prize - Medicine or Physiology - 1993

P.A. Sharp (Biology, MIT)

Proc. Natl. Acad. Sci. U.S.A. (1977) 74, 3171-5

studies on genetic structure of adenovirus 2

intronexon exon

Split Gene Structure

-eukaryotes from yeast to humans-90% of human genes-intron length highly variable-exon length ~200 nt-Dystrophin

mRNA

template DNA

?

Gene Structure Analysis by EM

Discovery of Split Genes (1977)

Voet and Voet Biochemistry

5’

mRNA

DNA

3’

I

II

IIIIV

V

VI

VII1 23 45 6

7

chicken ovalbumin

123 5 764

1 2 3 5 764

I II III IV V VI VII

7700 bp

1872 nucleotides

< 20% coding

Pre-mRNA Splicing

-splicing is nuclear (HeLa nuclear extracts)-requires Mg2+

-requires ATP-(is co-transcriptional)

IVS-E2

IVS

E1-IVS-E2

E1-E2

E1

Analysis of in vitro Splicing of 32P-Labeled pre-mRNA

A PPTOH2'

A

OH

A CAG-OH

step one

step two

CAGGU

CAGPPT

PPT

UG

UG

Pre-mRNA Splicing

Chemistry of Splicing

1st step 2nd step

P

OO

OG

OHO

O

-

OG

OH

O

O

OG

OHOH

O

P

OO

OG

OHO

O

-

OG

OHOH

O

OG

OH

O

O

5’ exon

5’ exon

5’ exon

5’ exon

3’ exon

3’ exon

OG

OHO

O

P

O

OO

OG

OHO

-

OA

OH

O

O

P

OO

OG

OHO

O

-

OA

O

O

OG

OHOH

O

O

Splicing Time Course

IVS-E2

IVS

E1-IVS-E2

Complex Formation in HeLa Extract

H

A

B

C

Discovery of the Spliceosome

Cell (1985) 42, 345-53

-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

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).

Cell (1999) 96, 375-87

snRNP Core Proteins

B,D1,D2,D3,E,F,G

U1

U2

U1

U1

U4

U4 U1

U1

U1

U1

U1U1

U2

U2

U4

U4

U2

U2

U4

U2

U2

U6

U6

U6

A

U5

U5

U5

U5

U5

U5

U5

-3'

-5' A

-5'

UG

-3'A AG

-5'

A

5'- GU -3'A AG

-3'A

U4/U6

U4/U6U4/U6

-5'

B1

C1

B2

C2

I

A

CC

ATP

ATP

ATP

b p

e x o n 15 '- e x o n 2 - 3 'G U A A G

P y

5 ' S S 3 ' S S

Pre-mRNA

A

- 3 '5 '- e x o n 1 e x o n 2

mRNA

A AG-5'

-3'

U6

-3'

U6

U4

U2

U6 U5

A AG

The spliceosome cycle

GU CAGPPTUACUAAC

5’ splice site 3’ splice site

poly-pyrimidine tract

branch region

Splicing Directed by Conserved Intron Sequences

Early Steps in Spliceosome AssemblyRecognition of the Pyrimidine Tract

. .

HeLa

poly-U

guanidine1 M KClΔ HeLa 2U AF

U2AF required for A complex

U2 Auxiliary Factor

-heterodimer, 65 kD, 35 kD subunits-U2AF65 required splicing factor-U2AF35, 3’ splice site

RNA BINDEFFECTORN C

U2AF65 Domain Structure

MSDFDEFERQLNENKQERDKENRHRKR S HSR S R S RDRKRR S R S RDRRNRDQR S ASRDRRRR S K-

U2AF65

Pyr tractBranch Region

.BBP

SR

AG

SR

U2AF65

U1snRNP

A

Bridged Commitment Complex

Py

EBCU2AF35

Bridged Commitment ComplexE Complex

Selection of 5’ Splice Site and Branch

GUAUGU5' exon

CAUUCAU1

••• ••

5' splice site

5'

UACUAC 3' exon

AUGAUGU2

• • • • • 5'

A

•34

UACUAAC 3' exon

3' splice siteBBP

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

GUAUGU5' exon

CAUUCAU1

••• ••

5' splice site

5'

GUAUGU5' exon

GACACAU6 47

• • •5'

3

Unusual Classes of Introns

AU ACA

“AT-AC”

minor spliceosome

U11 U1

U12 U2

U4atac/U6atac U4/U6

U5 common