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