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Nucleic Acids: Transcription and Translation - Lecture Notes, Exams of Topology

Site-directed mutagenesis. A. X. B. X. C. X. D. X. E. X. F. Replication. 1. Polymerases. 2. Fidelity a.Polymerase recognition b.Exonuclease.

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11/15/21
1
Lecture 25
(11/15/21)
Nucleic Acids
A. Nucleotides
B. Nucleic Acids
C. The 4 S’s
1. Size
2. Solubility
3. Shape
a. A-DNA
b. Z-DNA
c. Top ol og y
i. Packaging
ii. Supercoili ng
iii. Topoisomerases
4. Stability
a. Nucleotides
i. Tautomers
ii. Acid/base
b. Nucleic Acids
i. Chemistry
ii. Denaturati on
iii. Stabilit y
iv. Nucleases
D. Structure of the Information
1. Exceptions to flow
2. Structure
3. Levels of Control
E. Recombinant DNA: Biochemical Basis of Biotechnology
1. Restriction enzymes, DNA ligase
2. Vectors and Inserts to make recombinant DNA (rDNA)
3. Transformation of hosts
4. Selection of transformants
a. Selectable marker/gene
b. Distinguish empty plasmids
i. Loss of resistance
ii. Reporter gene
4. X
5. Expression
a. Special vectors
b. Fusion proteins
i. purification
ii. labeling
6. Site-directed mutagenesis
A. X
B. X
C. X
D. X
E. X
F. Replication
1. Polymerases
2. 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
3. Sequence determination
4. PCR
G. Transcription
1. Overview-mRNA structure
2. Process
3. RNA Polymerase
4. Fidelity
A. Translation
Reading: Ch26; 960-964
Ch27; 1006-
1013, 1018-
1024
Problems: Ch26; 1,2,6,
11, 17
Ch27; 5,8,9,11
NEXT
Reading: Ch27; 1015-
1018, 1028-
1036
Problems: Ch27; 6,7,10,
12,13,15,
16,18,19
Transcription & Translation
Tran sc rip ti on
Overview-mRNA structure
Process
RNA Polymerase
Fidelity
Tran sl ati on
Genetic Code
triplet
decyphering
tRNA
Structure
Anticodon
Acylation (charging)
Aminoacyl-tRNA Synthetases
Mechanism
Fidelity
Protein Biosynthesis
Overview
Process
Ribosome review
Peptidyl Transferase
Fidelity
pf3
pf4
pf5
pf8
pf9

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Lecture 25

Nucleic Acids

A. Nucleotides B. Nucleic Acids C. The 4 S’s

  1. Size
  2. Solubility
  3. Shape a. A-DNA b. Z-DNA c. Topology i. Packaging ii. Supercoiling iii. Topoisomerases
  4. Stability a. Nucleotides i. Tautomers ii. Acid/base b. Nucleic Acids i. Chemistry ii. Denaturation iii. Stability iv. Nucleases D. Structure of the Information
  5. Exceptions to flow
  6. Structure
  7. Levels of Control E. Recombinant DNA: Biochemical Basis of Biotechnology
  8. Restriction enzymes, DNA ligase
  9. Vectors and Inserts to make recombinant DNA (rDNA)
  10. Transformation of hosts
  11. Selection of transformants a. Selectable marker/gene b. Distinguish empty plasmids i. Loss of resistance ii. Reporter gene
  12. X
  13. Expression a. Special vectors b. Fusion proteins i. purification ii. labeling
  14. Site-directed mutagenesis

A. X

B. X

C. X

D. X

E. X

F. Replication

  1. Polymerases
  2. 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
  3. Sequence determination
  4. PCR G. Transcription
  5. Overview-mRNA structure
  6. Process
  7. RNA Polymerase
  8. 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

  1. 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