Macromolecular Machines - Introduction Biochemistry 4000 Dr. Ute Kothe
Dec 30, 2015
Macromolecular Machines- Introduction
Biochemistry 4000
Dr. Ute Kothe
Macromolecular Machines1. 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
Voet, Fig. 32-36
Large Subunit (50s) Small Subunit (30s)
E-site P-site A-sitetRNAs
Decoding center
E. coli Ribosome composition
Voet, Table 32-7
Translation Elongation Cycle
Voet, Fig. 32-48
1. Decoding /A-site binding
2. Peptide bond formation
3. Translocation
EF-Tu as aG protein switch
Ternary complexEF-Tu-GTP-aa-tRNA
EF-Tu-GTP
EF-Tu-GDP EF-Tu-EF-Ts
EF-Tu-GTP:• on/active• can bind aa-tRNA
EF-Tu-GDP:• off/inactive• can not bind aa-tRNA due to large scale domain rearrangements
EF-Ts: Guanine nucleotide exchange factor
Cryo-EM: Ribosome + EF-Tu-GTP-aa-tRNA
Stark et al., NSB 2002
13 Å resolution
Fitting of the crystal structures of ribosome and EF-Tu-GTP-aa-tRNA into the electron density
Ribosomal Decoding site
Voet, Fig. 32-64
Upon binding of the correct tRNA, A1492 and A1493 flip out and interact with the codon-anticodon duplex.
2nd position: A1492
codonanticodon
3rd position: G530
codonanticodon
1st position: A1493
codonanticodon
The decoding site: shape recognition
Voet, Fig. 32-63
Relaxed monitoring
of 3rd codon position
1st and 2nd position monitor geometry
of Watson-Crick basepair by measuring
Distances between riboses!
No specific interaction with bases!
Decoding Problem: difference in binding energies of cognate versus near-cognate (one mismatch) tRNAs not sufficient for efficient discrimination
DNA Polymerase I
Voet, Fig. 30-8
• 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
Taq Polymerase +/- substrate NTP
Voet, Fig. 30-9
Closed conformationIncoming nucleotide boundO helix (orange) closes over active site
Open conformationNo incoming nucleotideO 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.
Voet, Fig. 30-10
Most likely common catalytic mechanism for all DNA-Pol.:
• metal ion A (Mg2+) acitvates 3’OH of primer for nucleophilic attack on a-phosphate
• metal ion B (Mg2+) orients triposphate group for in-line attack and shields negative charges as well as additional charges in transition state
Animal DNA Polymerases
Voet, Table 30-5
RNA-PolymeraseBacteria: ’
Voet, Table 31-2
Taq RNA-Polymerase
Voet, Fig. 31-11 & 12
’– core enzyme– yellow & green – cyan’- pink - gray
Holoenzyme with subunit
Yeast RNA-Polymerase II
Voet, Fig 31-20
View from the right in left partNote similarity to bacterial RNA-Polymerase!
RNA-Pol II Elongation complex
Voet, Fig. 31-21
• “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”
Transcription cycle
Voet, Fig. 31-22
• 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
bent
Steitz, EMBO 2006
NucleotideAdditionCycle
T7 RNAPolymerase