Transcript Lec. 25 - Translation 3

The
Elongation
Cycle (in
prokaryotes)
Fig. 18.10
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; doesn’t
stop at unmodified (protected) residues
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
Locating the peptidyl transferase
on the large ribosomal subunit
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
“Yarus analogue”
Fig. 19.21 3rd ed.
50S subunit from
Haloarcula
X-ray crystal
structure
Yarus analogue
RNA - grey
proteins - gold
From Nissen et al., Science 289:920, 2000
Fig. 19.16
Nissen et al., Science 289: 920-930 (2000)
Active site: RNA + proteins
Active site: only proteins,
closest protein is at least
18 angstroms from the
phosphate of the Yarus
analogue.
Fig. 19.17
From Nissen et al., Science 289:920, 2000
Also Fig. 19.25 in Weaver
Evidence for rRNA as the PT
1.
2.
3.
4.
5.
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 loop.
Nearly all (99%) of the protein can be stripped from
the 50S subunit, and still 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.
• Are there any potential deficiencies with this
model or the data that support it?
• How could it be made stronger?
Fig.
19.28
tRNA Charging: The Second
Genetic Code
1. tRNA structure
2. the charging reaction
3. aminoacyl tRNA synthetases and
tRNA recognition
4. proofreading mechanism
Variable loop
General 3D structure of tRNA
Fig. 19.26
Fig.
19.25
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
tRNA Charging
• Occurs in two steps:
1. AA + ATP  Aminoacyl-AMP + PP
2. 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
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
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
Fig. 19.30
Class I - binds
from the D loop
side
Class II – binds
from the Variable
loop side
GlnRS – tRNAGln
(Class I)
AspRS-tRNAAsp
(Class II)
How is charging accuracy achieved,
given the structure of amino acids?
• Isoleucine tRNA synthetase (IleRS is Class I)
discriminates > 50,000-fold for Ile over
valine
• Ile and Val differ by only one methylene
group (Isoleucine has 1 more)
• Accuracy achieved by the IleRS having 2
active sites: 1st one activates most small
amino acids (to aa-AMP) and the 2nd one
hydrolyzes the aa-AMPs smaller than
Isoleucine (the editing site)
The double-sieve model for IleRS
Fig. 19.31