The role of rRNA in Peptide Bond Formation

1
The role of rRNA in Peptide Bond Formation

• The ribosome is a ribozyme.

Chapters 18.3, 19.1
2
The Elongation Cycle (in prokaryotes)
Fig. 18.10
3
Antibiotics that inhibit protein synthesis by
binding to ribosomes.
Chloramphenicol inhibits peptidyl transferase
(PT) activity!
Inhibits PT on 80S cytoplasmic ribosomes
Fig. 18.11
4
Puromycin resembles tyrosyl-tRNA, binds to the A
site, accepts peptide from peptidyl-tRNA
(catalyzed by PT).
Fig. 18.12
5
Fig. 18.22
Puromycin release assay for PT (1) load the P
site with labeled poly-Phe by adding poly U to a
translation mix, (2) add puromycin, (3) follow
puro-peptide released.
50S subunit contains the PT activity, which is
blocked by the antibiotics.
6
Fig. 18.24
The fragment assay uses CAACCA-f35SMet, which
binds to the P site, and puromycin, which binds
to the A site. PT activity indicated by formation
of fMet-puromycin.
Ribosomes (or 50S subunits) from E. coli (E) and
Thermus aquaticus (T) treated with protein
destroying agents still have peptidyl transferase
activity.
7
Fig. 18.25
99 deproteinized 50S subunits from T. aquaticus
have peptidyl transferase activity that is
inhibited by antibiotics and RNase T1.
8
Composition of the E. coli ribosome
50S subunit 23S 5S RNA 34 proteins 30S
subunit 16S RNA 21 proteins
Fig. 3.16
9
Gross anatomy of the E. coli ribosome.
head
platform
stalk
Central protuberance
ridge
platform
stalk
Fig. 19.5
10
Ribosome structure (cont.)
aa-tRNA in

• tRNAs occupy the canyon between the subunits
  they bind to both subunits
• 3 tRNA binding sites A (aminoacyl-tRNA), P
  (peptidyl-tRNA), and E (exit)

Short mRNA
A pre-Crystal Structure Model
tRNAs upside down!
11
The 50S subunit with the tRNAs bound in the E,P,A
sites
Modeled from crystal structures of the ribosomes
of Thermus thermophilus at 8 angstroms
resolution in the presence and absence of the
tRNAs.
Fig. 19.7
12
tRNAs bound mostly to RNA!
19.7a
13
Peptidyl-tRNA interacts with the 30S subunit at
the anticodon end, and with the 50S subunit at
the acceptor end.
Fig. 19.9
14
Footprinting drug binding sites on rRNA (Moazed
Noller)

• Analogous to footprinting a protein binding site
  on DNA or RNA
• Can map where antibiotics bind to rRNA in the
  ribosome
• Bound drug prevents chemical modification of the
  bases (use DMS for purines and CMCT for U)
• Modified bases cause reverse transcriptase to
  stop during primer extension doesnt stop at
  unmodified (protected) residues

15
Antibiotics that inhibit PT bind to a loop in
Domain V of 23S rRNA
Antibiotic footprints (circled bases)
PT loop
PT loop
PT loop
Antibiotic resistance mutations (circled bases)
PT loop peptidyl transferase loop
16
Locating the peptidyl transferase on the large
ribosomal subunit
17
2 analogues (b and c) that should bind to the
active site of PT on the large ribosomal subunit
(b) resembles the transition state formed
during the real reaction (a) (c) resembles a
substrate and docks into the A site
Fig. 19.21
Yarus analogue
18
50S subunit from Haloarcula X-ray crystal
structure
Yarus analogue
RNA - grey proteins - gold
From Nissen et al., Science 289920, 2000
Fig. 19.22
19
Nissen et al., Science 289 920-930 (2000)
20
Active site only proteins, closest protein is at
least 18 angstroms from the phosphate of the
Yarus analogue.
Active site RNA proteins
From Nissen et al., Science 289920, 2000 Also
Fig. 19.25 in Weaver
21
Evidence for rRNA as the PT

• No ribosomal proteins have been identified that
  have peptidyl transferase (PT) activity.
• Drugs (e.g., Chloramphenicol) that inhibit PT
  bind to the 23S rRNA, in the PT loop of Domain
  V.
• Mutations that provide resistance to the drugs
  that inhibit PT map to the same PT loop.
• Nearly all (99) of the protein can be stripped
  from the 50S subunit, and still get have PT
  activity.
• The X-ray crystal structure of the 50S subunit
  shows that only RNA chains (PT loop, etc.) are
  close enough to catalyze a reaction.

22

• Are there any potential deficiencies with this
  model or the data that support it?
• How could it be made stronger?

23
tRNA Charging The Second Genetic Code

• tRNA structure
• the charging reaction
• aminoacyl tRNA synthetases and tRNA recognition
• proofreading mechanism

24
General 3D structure of tRNA
Fig. 19.33
25
Amino acids are attached to the 3 terminal nt of
tRNAs (adenosine), via the 3 or 2 OH group.
3 term. A
Amino acid portion
26
Recognition of aminoacyl-tRNAs by the ribosome
Fig. 19.37

• Ribosomes only recognize the tRNA part, not the
  amino acid.
• Therefore, tRNA charging needs to be very
  accurate.

27
tRNA Charging

• Occurs in two steps
• AA ATP ? Aminoacyl-AMP PP
• Aminoacyl-AMP tRNA ?Aminoacyl-tRNA AMP
• Catalyzed by Aminoacyl-tRNA synthetases
• Cells must have at least 20 aminoacyl-tRNA
  synthetases, one for each amino acid

28
Recognition of tRNAs by Aminoacyl-tRNA
synthetases the Second Genetic Code

• Aminoacyl-tRNA synthetases recognize mainly the
  acceptor stem and the anticodon.

From Voet and Voet, Biochemistry
29
Aminoacyl-tRNA synthetases (cont.)

• Diverse group of enzymes despite recognizing
  fairly similar substrates
• Not well conserved, however there are 2 main
  classes
• Class I (aminoacylate the 2 OH)
• Class II (aminoacylate the 3 OH)
• Each class has the same 10 members in all
  organisms
• The classes bind tRNA somewhat differently, but
  both bind to the acceptor stem and the anticodon
  loop

30
How is charging accuracy achieved, given the
structure of amino acids?

• Isoleucine tRNA synthetase (IleRS) discriminates
  gt 50,000-fold for Ile over valine
• yet Ile and Val differ by only one methylene
  group
• accuracy achieved by the IleRS having 2 active
  sites one that charges tRNA and one that
  hydrolyzes mischarged aminoacyl-tRNAs (the
  editing site)

31
The double-sieve model for IleRS
Fig. 19.41