The Ribosome

1
The Ribosome

• The Central Dogma
• This states that once information has passed
  into protein it cannot get out again.
• In more detail, the transfer of information from
  nucleic acid to nucleic acid, or from nucleic
  acid to protein may be possible, but the transfer
  from protein to protein, or from protein to
  nucleic acid is impossible.
• -Francis Crick, On Protein Synthesis, 1957

2
The Central Dogma of Molecular Biology
transcription
translation
DNA RNA protein
3
RNA and DNA can both form a double helix
Base complementarity is the basis for genetic
replication. Watson-Crick base pairing
RNA (A-form)
DNA (B-form)
4
The Central Dogma of Molecular Biology
transcription
translation
DNA RNA protein
Why Dogma?
My mind was, that a dogma was an idea for which
there was no reasonable evidence, you see? -F.
Crick
5
Translation The process of decoding messenger
RNA (mRNA) to synthesize proteins Exclusively
catalyzed by the Ribosome
amino acid moiety
6
The Prokaryotic (70S) Ribosome Components
70S
50S
30S
Total Molecular Weight 2,500,000 Daltons
7
Structure of transfer RNA (tRNA)
2 structure
8
The 70S Ribosome binds A-, P-, and E- site tRNAs
50S 30S
tRNAs
OH
3-CCA end
codonanticodon pairs
mRNA
E P A
E (exit) site binds deacylated tRNA P
(peptidyl) site binds peptidyl-tRNA A
(aminoacyl) site binds to aminoacylated tRNA
9
A comment on resolution...
The models that we construct to support our data
are limited the the inherent resolution of the
data itself.
10
(No Transcript)
11
(No Transcript)
12
Progress Towards the Structure of the Ribosome
1955
microsomes
1960
two subunits
High Resolution (8-15 Å) Cryo-electon microscopy
1995
13
Electron Density of the 70S Ribosome at 5.5 Å
2001
14
The Ribosome is Coming into Focus...
increasing resolution over 50 years.
15
Et tu, Brute?
16
The ribosome is a ribozyme!
17
(No Transcript)
18
The Life Cycle of Prokaryotic Translation Phase
1 Initiation initiation factors IF-1, IF-2,
IF-3 initiator tRNAfMet, mRNA (SD
sequence) Phase II Elongation elongation
factors EF-TutRNAGTP, EF-GGTP Phase III
Termination release factors RF-1 (or RF-2),
RF-3 Phase IV Recycling ribosome recycling
factor (RRF), EF-G, IF-3
19
Phase I initiation
20
Proposed structures of initiation factors bound
to 30S
21
Consensus SD sequence - signals translation
initiation
22
(No Transcript)
23
A-, P- and E- tRNAs bind at the subunit interface
24
The Elongation Phase of Translation
50S 30S
mRNA
E P A
25
(No Transcript)
26
Why 2 Subunits?
50S Responsible for binding the 3CCA end
of each tRNA Has peptidyl transferase
activity 30S Binds to mRNA Responsible for
proper codonanticodon interactions
(maintains the reading frame fidelity) 50S
and 30S might move with respect to each other to
move tRNAs through the ribosome (translocation).
27
hybrid states model of translocation
50S 30S
Moazed and Noller, Nature (1989)
28
Elongation Factors EF-Tu (w/tRNA) and EF-G
29
The ribosome can discriminate between cognate and
near cognate tRNAmRNA pairs by use of 3º RNA
structural interactions The A-minor Motif
30
CryoEM Cryogenic Electron Microscopic
Reconstructions This technique has shown the 70S
ribosome in different functional conformations at
medium-to-low resolutions. Complex
I deacylated tRNA Complex II deacylated tRNA
EF-G/GDPNP
31
(No Transcript)
32
tRNA movements of the pre-translocation complex
33
The PTC ribosome active site!
34
Proposed Mechanism of Termination and Recycling
50S 30S
mRNA
E P A
35
RF1 and RF2 conformational changes and tRNA
mimicry
36
Release factors mimic the anticodon loop at the
decoding site of the 30S subunit
37
Release factors also coordinate at the PTC to
catalyze tRNA-peptidyl bond cleavage (GGQ motif)
38
Release factor RF3 binds to the ribosome
similarly to EF-G (both are GTPases) - catalyzes
release of RF1/2.
39
(No Transcript)
40
RRF binds across the subunit interface no
P-site tRNA!
41
Thus far, we have seen many structures, both of
high and low resolution, that describe different
functional states of the ribosome.... This is
only part of the story! the ribosome is a
dynamic translation machine! New methods of
experimental query single molecule measurements
42
(No Transcript)
43
(No Transcript)
44
(No Transcript)
45
Amazingly, antibiotics inhibit the ribosome in
virtually every step in the elongation phase of
translation EF-Tu release kirromycin A-si
te tRNA binding tetracycline tRNA
miscoding kanamycin, neomycin A-site tRNA
release paromomycin, hygromycin B Classical
state conformation spectinomycin Hybrid state
conformation viomycin peptidyl transferase
activity sparsomycin, puromycin chloram
phenicol EF-G binding thiostrepton EF-G
Release fusidic acid mRNA binding/movement
kasugamycin
46
WARNING
The movie you are about to see is what happens
when scientists are given the freedom to express
their love for the ribosome....
47
(No Transcript)
48
(No Transcript)
49
(No Transcript)