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Site-directed mutagenesis. A. X. B. X. C. X. D. X. E. X. F. Replication. 1. Polymerases. 2. Fidelity a.Polymerase recognition b.Exonuclease.
Typology: Exams
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A. Nucleotides B. Nucleic Acids C. The 4 S’s
F. Replication
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
prokaryotes
eukaryotes Start of Translation
End of Translation
OPEN READING FRAME 5’-UTR (^) (ORF)
Shine-Delgarno 5’-cap
3’-Poly(A)
Stop Codon
AAGGAGGU
…AAAAAAAAAA
poly(A) polymerase (PAP)
poly(A)-binding protein (PABP II)
Coding strand
Template strand
Actinomycin D-DNA complex PDBid 1DSC
Inhibits elongation by intercalating
Inhibits elongation at first phosphodiester bond
Inhibits EUKARYOTIC RNAPs (only I & III)
These subunits make up the core complex:
(a 2 bb’ws)
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
Closed conformation
Also, the b’ homolog has a disordered CTD that is phosphorylated In going from Initiation to elongation
Open conformation
Gold = hydrophobic amino acids; pyrimidine at second position
Polar amino acids (blue = basic; red = acidic; purple = uncharged polar) have purine at second position
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
Translation: The Genetic Code
Result: UUU=Phe, AAA=Lys, CCC=Pro, GGG=Gly
Result: 50/64 determined