Macromolecular Machines
- Introduction
Biochemistry 4000
Dr. Ute Kothe
Macromolecular Machines
1. DNA Polymerase – DNA replication, Voet chapter 30
error rate: 10-7
2. RNA-Polymerase – Transcription, Voet chapter 31
error rate: 10-3
3. Ribosome – Translation, Voet chapter 32
error rate: 10-3 - 10-4
Unifying question:
How do all these machines achieve high accuracy in
duplication and expression of genetic information?
In each case, Watson-Crick base-pairs have to be selected
with high accuracy against non-Watson-Crick base pairs.
The Ribosome
Large Subunit (50s)
Small Subunit (30s)
Decoding center
E-site P-site A-site
tRNAs
Voet, Fig. 32-36
E. coli Ribosome composition
Voet, Table 32-7
Translation Elongation Cycle
1. Decoding /
A-site binding
3. Translocation
2. Peptide bond
formation
Voet, Fig. 32-48
EF-Tu as a
G protein switch
EF-Tu-GTP
Ternary complex
EF-Tu-GTP-aa-tRNA
EF-Tu-GTP:
• on/active
• can bind aa-tRNA
EF-Tu-GDP:
• off/inactive
• can not bind aatRNA due to large
scale domain
rearrangements
EF-Ts:
Guanine nucleotide
exchange factor
EF-Tu-GDP
EF-Tu-EF-Ts
Cryo-EM:
Ribosome + EF-Tu-GTP-aa-tRNA
Fitting of the crystal
structures of
ribosome and EFTu-GTP-aa-tRNA
into the electron
density
13 Å resolution
Stark et al., NSB 2002
Ribosomal Decoding site
Upon binding of the correct tRNA, A1492 and A1493 flip
out and interact with the codon-anticodon duplex.
Voet, Fig. 32-64
The decoding site: shape recognition
1st position: A1493
anticodon
codon
2nd position: A1492
3rd position: G530
anticodon
anticodon
codon
1st and 2nd position monitor geometry
of Watson-Crick basepair by measuring
Distances between riboses!
No specific interaction with bases!
codon
Relaxed monitoring
of 3rd codon position
Decoding Problem: difference in binding energies of cognate
versus near-cognate (one mismatch) tRNAs not sufficient for
efficient discrimination
Voet, Fig. 32-63
DNA Polymerase I
• bacterial DNA-Polymerase, single polypeptide, highly processive
• Proofreading ability: 3’-5’ exonuclease, 5’-3’ exonuclease
• Klenow fragment: C-terminal fragment with polymerase & 3’5’exonuclease activity
Voet, Fig. 30-8
Taq Polymerase +/- substrate NTP
Voet, Fig. 30-9
Closed conformation
Incoming nucleotide bound
O helix (orange) closes over active site
Open conformation
No incoming nucleotide
O helix away from active site
Recognition of incoming dNTP
• Recognition of shape of base pair
independent of hydrogen bonding
properties
• Conserved Tyr stacks on template base
• Last 3 nucleotides in A-DNA
conformation with wider minor
groove which is monitored by amino
acids for N3 of purines and O2 of
pyrimidines
• 2,4-Difluorotoluene (F) can be inserted
instead of thymine (T) by DNA-Pol I
(isosteric, but can not accept hydrogen
bonds)
Catalytic Mechanism of DNA-Pol.
Most likely common catalytic
mechanism for all DNA-Pol.:
• metal ion A (Mg2+) acitvates 3’OH of
primer for nucleophilic attack on aphosphate
• metal ion B (Mg2+) orients
triposphate group for in-line attack
and shields negative charges as well
as additional charges in transition
state
Voet, Fig. 30-10
Animal DNA Polymerases
Voet, Table 30-5
RNA-Polymerase
Bacteria: a2bb’ws
Voet, Table 31-2
Taq RNA-Polymerase
Holoenzyme
with s subunit
a2bb’w – core enzyme
a – yellow & green
b – cyan
b’- pink
w - gray
Voet, Fig. 31-11 & 12
Yeast RNA-Polymerase II
Note similarity to bacterial
RNA-Polymerase!
View from the right in left part
Voet, Fig 31-20
RNA-Pol II Elongation complex
• “clamp” swings over DNA to trap it, ensures high processivity
• unwound template strand make 90° turn after active site due to “wall”
• active site accessible through funnel for new NTPs
• sequence-independent contacts of enzyme with sugar-phosphate backbone
• DNA-RNA hybrid helix is disrupted by “rudder”
Voet, Fig. 31-21
Transcription cycle
bent
• Highly conserved “bridge helix” contects two pincers forming the enzyme’s
cleft
• Bridge helix nonspecifically contacts template DNA at +1 position
• Straight in RNA-Pol II, bent in Taq RNA-Pol.
 Might alternate between straight and bent conformation moving by 3- 4 A
 Might push paired nucleotide at position +1 to position -1 during
translocation
Voet, Fig. 31-22
T7 RNA
Polymerase
Nucleotide
Addition
Cycle
Steitz, EMBO 2006