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Lecture 25
Nucleic Acids
A. Nucleotides B. Nucleic Acids C. The 4 S’s
- Size
- Solubility
- Shape a. A-DNA b. Z-DNA c. Topology i. Packaging ii. Supercoiling iii. Topoisomerases
- Stability a. Nucleotides i. Tautomers ii. Acid/base b. Nucleic Acids i. Chemistry ii. Denaturation iii. Stability iv. Nucleases D. Structure of the Information
- Exceptions to flow
- Structure
- Levels of Control E. Recombinant DNA: Biochemical Basis of Biotechnology
- Restriction enzymes, DNA ligase
- Vectors and Inserts to make recombinant DNA (rDNA)
- Transformation of hosts
- Selection of transformants a. Selectable marker/gene b. Distinguish empty plasmids i. Loss of resistance ii. Reporter gene
- X
- Expression a. Special vectors b. Fusion proteins i. purification ii. labeling
- Site-directed mutagenesis
A. X
B. X
C. X
D. X
E. X
F. Replication
- Polymerases
- Fidelity a.Polymerase recognition b.Exonuclease i. 3’à5’ ii. 5’à3’ c.Mis-match repair d.Post-replication repair i. Direct reversal ii. Base excision iii. Nucleotide excision
- Sequence determination
- PCR G. Transcription
- Overview-mRNA structure
- Process
- RNA Polymerase
- Fidelity A. Translation
- Reading: Ch26; 960- Ch27; 1006- 1013, 1018- 1024
- Problems: Ch26; 1,2,6, 11, Ch27; 5,8,9, NEXT
- Reading: Ch27; 1015- 1018, 1028- 1036
- Problems: Ch27; 6,7,10, 12,13,15, 16,18,
Transcription & Translation
Transcription Overview-mRNA structure Process RNA Polymerase Fidelity Translation Genetic Code triplet decyphering tRNA Structure Anticodon Acylation (charging) Aminoacyl-tRNA Synthetases Mechanism Fidelity Protein Biosynthesis Overview Process Ribosome review Peptidyl Transferase Fidelity
Nucleic acid function: Central Dogma
Transcription
mRNA structure
5’ cap and 3’ polyA tail
prokaryotes
eukaryotes Start of Translation
End of Translation
AUG
OPEN READING FRAME 5’-UTR (^) (ORF)
3’-UTR
Shine-Delgarno 5’-cap
3’-Poly(A)
Stop Codon
Transcription
AAGGAGGU
…AAAAAAAAAA
poly(A) polymerase (PAP)
poly(A)-binding protein (PABP II)
RNAP Complexed With Promoter
Coding strand
Template strand
Inhibitors of Transcription
Actinomycin D-DNA complex PDBid 1DSC
Transcription
Transcription
Mechanism
Inhibits elongation by intercalating
Inhibits elongation at first phosphodiester bond
Inhibits EUKARYOTIC RNAPs (only I & III)
RNA Polymerase Structure RNA pol^ is a multi-subunit enzyme
These subunits make up the core complex:
- two α subunits make non-specific contacts with DNA for positioning
- the β and β’ subunits catalyze the addition of ribonucleotides to the growing chain
- the ω subunit acts to stabilize the complex
(a 2 bb’ws)
Transcription
Nascent RNA
Subunit Gene MW # Role
a 2 rpoA^^34 2 Non-specific DNA binding b rpoB^^150 1 Polymerase b’ rpoC^^155 1 Non-specific DNA binding& polymerase
w rpoZ^^10 1 Zn2+^ binding s rpoD^^70 1 Promoter recognition
Template DNA Coding strand DNA RNA•DNA duplex is in the A-form It uses the same mechanism for correct W-C bp and fidelity Therefore, error rate is the same 1/10,
Rate is ~ 50 base/sec Processivity is ~2000 bp
No 3’à5’ exonuclease proofreading
Coding strand
Template strand
Yeast RNA polym erasePDBid 1I
Transcription
Eukaryotic RNA Polymerase II Conformations
Closed conformation
Also, the b’ homolog has a disordered CTD that is phosphorylated In going from Initiation to elongation
Open conformation
Nucleic acid function: Central Dogma
Transcription & Translation
Recall: Genetic Code is Degenerate & Nonrandom
Gold = hydrophobic amino acids; pyrimidine at second position
Polar amino acids (blue = basic; red = acidic; purple = uncharged polar) have purine at second position
Translation: The Genetic Code
How was the Triplet
code discovered?
THE BIG RED FOX ATE THE EGG
THE IGR EDF OXA TET HEE GG
THE IGR EDX FOX ATE THE EGG
Translation: The Genetic Code
If one base is deleted:
If one base is then inserted:
ORFs X
The regain of function for the triple mutant told Brenner and Crick that it was a triplet code, uninterrupted.
How was the code deciphered?
Brenner & Crick Experiment:
Nirenberg (NIH)
Key Developments:
**1. Chemical synthesis of nucleic acids
- in vitro protein synthesis**
Translation: The Genetic Code
1. First codons used Polynucleotide
Phosphorylase (NDP ⇌ RNA + P i ) to
make RNA in vitro: poly-A, poly-C,
etc.
2. Chemical synthesis of defined triplets.
- Use ribosomes and charged tRNA with
different radioactive amino acids
- Mix and filtrate - only those amino
acids with correct tRNA to
complementary “mRNA” will complex
with the ribosome
Result: UUU=Phe, AAA=Lys, CCC=Pro, GGG=Gly
Result: 50/64 determined
