Ribosome Structure and Assembly

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Ribosome Structure and Assembly
1. Introduction The Process of Translation
2. Structures of the Ribosome and Subunits
3. Ribosome Assembly (30S)
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1st step Initiation
T. Terry, U. Conn
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2nd step Elongation
T. Terry, U. Conn
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Last step Termination
T. Terry, U. Conn
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Ribosome Structure and Assembly
1. Introduction The Process of Translation
2. Structures of the Ribosome and Subunits
- Predictions from sequence analysis
- x-ray crystallography determinations
3. Ribosome Assembly (30S)
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Early Evaluation of the Shape of the Ribosome by
EM
First EM images in 1950s
Molecules in different orientations combined to
create models
Proteins localized by binding antibodies
Most early and later structural work on
prokaryotic ribosomes
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Comparative Sequence Analysis Used to Predict
Most of the Base Pairs
Method first applied to tRNA (1970) and 5S rRNA
(1975)
First secondary structure predicted in 1980
(Woese, Noller, and colleagues)
16S rRNA can be divided into subdomains. Much
of secondary structure is local
60 of nucleotides are base-paired
1500 nt
H. Noller lab web page
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3000 nt
H. Noller lab web page
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Identification of Secondary Structures By
Sequence Analysis
Sequence analysis can predict secondary
structure by finding base-pairing potential
When multiple related sequences are available,
covariation provides additional evidence for
pairing
This figure happens to show a riboswitch (more
next Tuesday on that), but the same methods were
used to deduce secondary structures of the
rRNAs. Notice the pseudoknot in this picture and
sequence above
Corbino et al, Genome Biology 2005, 6R70
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Comparative Sequence Analysis Used to Predict
Most of the Base Pairs
97 of predicted base pairs were present in
crystal structures
75 of base pairs in crystal structures were
predicted. Others were not detectable because
they do not vary between sequences.
Some tertiary interactions show covariation and
can therefore be predicted
Tetraloop-receptor interactions
Many tertiary contacts mediated by A nucleotides
Gutell et al, Curr. Opin. Struct. Biol. (2002)
12, 301-310.
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Crystal Structure of tRNA
First molecular details of higher-order RNA
structure, demonstrated tertiary contacts
Two groups (A. Rich and A. Klug) published
structures in 1974. First author on Klugs work
was Jon Robertus (UT Biochemistry).
Fig. 19.26
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High-resolution Structure of a 16S RNP Domain
First atomic resolution view of ribosomal
subdomain
Structure included 104-nt RNA and 3 proteins
S15, S6, and S18
Suggests hierarchical assembly of RNA-protein
structure
Agalarov et al, Science (2000) 288, 107-112
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70S Ribosome of Thermus thermophilus
Noller and colleagues, 5.5 Å, 2001
Structure includes 30S, 50S, associated
proteins, and three tRNA molecules bound in A,
P, and E sites
16S rRNA, cyan 30S proteins, blue 23S
rRNA, gray, 5S rRNA, dark blue 50S
proteins, purple
Showed that core and interface are dominated by
RNA, not protein
Ribosome ripped apart to expose tRNAs
Fig. 19.1
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Codon-anticodon Base Pairing
Bend in mRNA between A and P sites allows
adjacent tRNAs to bind to consecutive codons in
proper orientations for peptidyl transfer
Fig. 19.2
Stereo image Try to see the image in 3D by
looking at your book
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Bridges Between the 30S and 50S Subunits
Most of the intersubunit bridges are RNA
16S and 23S rRNA, gray 5S rRNA, blue Proteins,
light blue RNA-RNA bridges, pink Protein-protein
and protein-RNA bridges, yellow
All bridges near tRNAs are RNA
Translocation requires large movements some
bridges may be dynamic
Fig. 19.3
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Ribosome Schematic Based on Structural Information
Large cavity between subunits to accommodate
the three tRNAs
Fig. 19.4
tRNAs interact with 30S subunit through
anticodon ends and bind to mRNA, also bound to 30S
tRNAs interact with 50S through acceptor stems.
This is where peptidyl transfer happens
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Structure of the 30S Subunit
3D structure (same colors)
Secondary structure
Central domain
Central domain structure remains intact
Overall, tertiary arrangement dominated by RNA
Fig. 19.8
Fig. 19.9
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50S Structure
Monolithic RNA, not modular
Most of protein mass on or near surface
Portions of proteins toward middle are in
unprecedented, unfolded conformations threaded
through RNA
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50S Structure Shows No Proteins Near Active Site
For Peptidyl Transfer
The ribosome is a ribozyme
Proteins snake toward, but not into, active site
Fig. 19.17
Fig. 19.16
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Ribosome Structure and Assembly
1. Introduction The Process of Translation
2. Structures of the Ribosome and Subunits
3. Ribosome Assembly (30S)
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Processing of the rRNA Precursor
E. coli has seven operons (rrn) that encode
rRNAs
Cleavage events give rise to processed rRNAs
16S
23S
5S
Fig. 16.4
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2D Gel Electrophoresis Shows Protein Components
Method first used to ribosomal proteins in 1970
30S 21 proteins
Ribosomes isolated, then proteins separated on
gel
Here, two dimensions were native PAGE at two
different pH values and acrylamide concentrations
Fig. 19.5
Eukaryotic ribosome is more complex - 40S
(30S equivalent) has 30 proteins - 60S (50S
equivalent) has 40 proteins and 3 RNAs (28S,
5.8S, and 5S)
50S 34 proteins
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Binding of Some 30S Proteins Is Necessary For
Binding of Others
30S reconstitution demonstrated by Nomura and
colleagues (late 1960s)
Added back proteins in different orders to
build up assembly pathway
3H-labeled S12
A. S4, S7, S8, S13, S16, S20 B. S4, S8, S16,
S17 C. All except S12
Sucrose gradient ultracentrifugation
Fig. 19.6
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The Nomura 30S Assembly Map
Proteins separated into primary, secondary, and
tertiary binders
S15, S17, S4, S8, S20, S13, and S7 are primary
binders
In general, proteins lower down on the map are
on outside of 30S particle.
Suggests similarity between thermodynamic
pathway outlined here and kinetic pathway in vivo
Much less known about 50S assembly
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Kinetics of 30S Reconstitution
Complete 30S subunit formation assayed by
activity in translation assay
Very strong temperature dependence for
formation kinetics
Proteins below dashed line in fig. 19.7
(previous slide) are not bound stably in RI
intermediate
Traub and Nomura, J. Mol. Biol. (1969), 40,
391-413
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Mass Spectrometry Approach To Follow Association
of Individual Proteins With 16S rRNA
Assembly initiated with 15N-labeled proteins,
then chased with unlabeled proteins
Extent of binding of each protein at time of
chase measured by mass spectrometry
Primary binders from Nomura map mostly bind fast
Binding is faster in general for proteins that
bind closer to 5 end
Colors represent relative binding rates
Talkington and Williamson, Nature (2005) 438,
628-632
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Are Molecular Chaperones Involved in Ribosome
Assembly?
Three of five DEAD-box proteins in E. coli are
implicated in ribosome assembly
The Hsp70 protein chaperone (DnaK) has been
implicated also
Numerous small RNAs (snoRNAs) are required for
assembly of the eukaryotic ribosome, and these
RNAs bind transiently to regions within the
rRNAs. Some of them direct modifications, but
some could also function as chaperones.
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Key Points
1. Prokaryotic ribosomes are composed of two
subunits, the 50S and the 30S. The 50S subunit
includes two rRNAs, the 23S and 5S rRNAs, and 34
proteins. The 30S subunit includes one rRNA, the
16S, and 21 proteins.
2. Recent structural analyses have revolutionized
our understanding of the ribosome. Most of the
base pairs and many tertiary contacts were
predicted correctly by comparative analysis, but
the structures reveal molecular details,
interactions with proteins, and many features
that were not predictable.
3. The 30S subunit can be reconstituted from pure
16S RNA and proteins. This process is thought to
involve hierarchical steps of RNA folding and
protein binding, but many features remain poorly
understood.
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