Locating the peptidyl transferase on the large ribosomal subunit

1
Locating the peptidyl transferase on the large
ribosomal subunit
2
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
3
50S subunit from Haloarcula X-ray crystal
structure
Yarus analogue
RNA - grey proteins - gold
From Nissen et al., Science 289920, 2000
Fig. 19.22
4
Nissen et al., Science 289 920-930 (2000)
5
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
6
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.

7

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

8
tRNA Charging The Second Genetic Code

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

9
General 3D structure of tRNA
Fig. 19.33
10
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
11
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.

12
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

13
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
14
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

15
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)

16
The double-sieve model for IleRS
Fig. 19.41