Biochemistry 430/530 Andrew MacMillan andrew.macmillan@ualberta MedSci 4-39 (by appointment)

Biochemistry 430/530Andrew [email protected] 4-39 (by appointment)492-3813

http://www2.biochem.ualberta.ca/MacMillanLab/

“Course Notes”

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Medicine/Physiology Chemistry

Szostak, Greider, Blackburn Steitz, Ramakrishnan, Yonath

1989 Sidney Altman, Thomas R. Cech2006 Andrew Z. Fire, Craig C. Mello1993 Richard J. Roberts, Phillip A. Sharp

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Classical Gene Regulation

1) RNA Structure/Reactivity (1)2) Ribozymes/Riboswitches(2)3) pre-mRNA Splicing (3)4) RNA Degradation/RNAi(2)

RNA

information

function

structure

Central Dogma

DNA RNA Protein

Central Dogma

DNA RNA Protein

DNA RNA Protein

Central Dogma

ProcessingprecursorRNAspre-mRNApre-tRNApre-rRNApre-snoRNAcap formationend formationsplicingeditingchemical modification(rare bases)RNAi (siRNA, miRNA)

Hammerhead Ribozyme

UG

CU

C

CC

GC

GC

GG

UC

UGA

GUCCG

GAC

ACA

GG

AA

5'3'

XA

5' 3'

3'5'

Ribosome - Large SubunitH. marismortui

Science (2000) 289:920-30

-2833 nt-27 proteins

Chemistry Review!

El LG Nu ElNu

LG+

H A H BB

A+

M2+

+ R-O-H R-O-HM2+

Acid/Base - Bronsted Base

Acid/Base - Lewis Acid

Nucleophilic Substitution

RNA Structure and Reactivity

N

NN

N N

NN

N

N

N

N

N

N

NH

O

H

OH

N

OO O

H

O

NH

HN

H

H

H

G A

U 5-Me-U C

OO

O O

Base

PO

P

O

HO

O O

O

1

23

4

567

8

9

3

216

54

1’2’3’

4’

5’

Nucleic AcidsChemical

Functionalities-Hydrogen Bonding-Metal Coordination-Acid/Base-Nucleophilic Reactions

Hydrogen Bonding

N

NN

N

NN

HN

O

H

O

NH

H

H

G

C

N

NN

N

NN

HN

O

H

O

NH

H

H

G

C

D

A

~3-5 kcal/mol

Hydrogen Bonding

N

NN

N

NN

HN

O

H

O

NH

H

H

G

C

N

NN

N

NN

HN

O

H

O

NH

H

H

G

C

D

A

~3-5 kcal/mol(excluding entropy - how much is this worth?)

Metal Coordination

Na+, K+, Mg2+, Mn2+

O

POO

O

M2+

-

G5

G16.2

Acid/Base

N

N

HN

O

H

H+

N

N

HN

O

H

pKa ~ 4

O

OH OH

NO

O OH

N

P

O

O

O-

Acid/Base

N

N

HN

O

H

H+

N

N

HN

O

H

pKa ~ 4

O

OH OH

N

pKa ~ 12

O

O OH

N

P

O

O

O-

pKa ~ 14

Nucleophilic Reactions I

N

NN

N

H

O

NH

H

S

O

OCH3CH3O

O

N

NN

N

O

NH

H

N

NN

N

H

O

NH

H

CH3+

CH3

Nucleophilic Reactions I

N

NN

N

H

O

NH

H

S

O

OCH3CH3O

O

N

NN

N

O

NH

H

N

NN

N

H

O

NH

H

CH3+

CH3

Nucleophilic Reactions II

O

OHO

P

O

O

O-

NO

OO

N

P

O O-

Minor

Major

B-Form DNA

Dickerson and coworkers

A-Form RNA

Minor

Major

N

NN

N

N

NN

N

NN

NN

H

O

H N

O

O

H

O

NH

HN

H

H

H

G

A

U

C

N

NN

N

N

N

H

O

O

HN

H

A

U

Base-Pairing in RNA

Watson-Crick Hoogsteen

N

NN

N

N

NN

N

NN

NN

H

O

H N

O

O

H

O

NH

HN

H

H

H

G

A

U

C

N

NN

N

N

N

H

O

O

HN

H

A

U

Base-Pairing in RNA

Watson-Crick Hoogsteen

Major

Minor

A+-C in Double-Stranded RNA

J. Mol. Biol. 283, 977 (1998)

Higher Order Structure in Nucleic AcidsFrom Non-WC Base-Pairs

N

NN

N

H

O

NH

H

G

N

N

NN

H

O

N

H

H

N

N N

N

H

O

NH

H

N

N

N N

H

O

N

H

H

G

G

G

M+