Macromolecular Machines- Summary of Publications

Biochemistry 4000

Dr. Ute Kothe

Kinetic Mechanism of A-Site Binding

Proofreading

Initial Selection

Accuracy in protein synthesis

Proofreading

Initial Selection1. Kinetic proofreading:

Two selection phases

separated by irreversible

step (GTP hydrolysis =

energy investment)

Multiplies accuracy

2. Induced Fit:

Forward steps significantly

Faster for correct

substrate,Increases speed

and accuracy

3. Stability difference

Back steps faster for

incorrect substrate

RNA-Polymerase

• can use RNA template

RdRP activity (RNA-dependent RNA Polymerase activity)

• Assay to check for elongation of RNA:

• fluorescently labeled RNA

• addition of selected NTPs which are complementary to template

• analysis of fluorescent, elongated RNA product on high-percentage gel (single nucleotide resolution)

RNA-Polymerase• RNA(product)-RNA(template) duplex is an A-type helix

• usual RNA(product)-DNA(template) duplex is A-type helix as well

Both fit into RNA-Pol exit cleft

Significance:

• Evolution: RNA-Polymerase might have evolved shortly after RNA world to replicate RNA which still contained the genetic information

• Hepatitis Delta Virus: RNA-Pol + TFIIS might cleave RNA genome to allow for direct replication by RNA-Pol. (circumventing the need for reverse transcription to generate DNA)

DNA-Polymerase

1. Insertion site: incoming nucleotide pairs with template base n2. Catalytic site: 3’hydroxyl of primer strand, 2 catalytic Mg2+ ion3. Pre-insertion site: template base N prior to incorporation 4. Post-insertion site: grwoing end of duplex DNA (new base-pair n-

1 after translocation)5. DNA duplex binding region: binding of 4 base-pair (n-2, ..., n-5)

duplex to polymerase

DNA-PolymeraseMismatch-Induced Disruptions:- depending on type of mismatch

1. Disruption of template strand and pre-insertion site ***

2. Disruption of primer strand and assembly of catalytic site

3. Disruption of template and primer strands

4. Fraying of DNA at insertion site

*** G-T mismatch in postinsertion site AND G-T mismatches in position n-2 and n-3

“Short term memory”: disruption of active site for at least 3 rounds of elongation

1.

4.

3.

2.