The Central Dogma (Francis Crick, 1958)

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The Central Dogma(Francis Crick, 1958)
(Transcription)
(Translation) DNA ? RNA ?
Protein (Gene/Genotype)
(Phenotype) An informational process between the
genetic material (genotype) and the protein
(phenotype)
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Classes of RNA for Transcription and Translation

• Informational RNA (intermediate in the process of
  decoding genes into polypeptides)
• Messenger RNA (mRNA)
• Functional RNAs (never translated into proteins,
  serve other roles)
• Transfer RNAs (tRNA)
• Transport amino acids to mRNA and new protein
• Ribosomal RNAs (rRNA)
• Combine with an array of proteins to form
  ribosomes platform for protein synthesis
• Small nuclear RNAs (snRNA)
• Take part in the splicing of primary transcripts
  in eukaryotes
• Small cytoplasmic RNAs (scRNA)
• Direct protein traffic in eukaryotic cells
• Micro RNAs (miRNA)
• Inhibits translation and induces degradation of
  complementary mRNA

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RNA nucleotide sequences are complementary to DNA
molecules
New RNA is synthesized 5 to 3 and antiparallel
to the template
DNA template
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Only one strand of the DNA acts as a template for
transcription
The template strand can be different for
different genes But. For each gene only one
strand of DNA serve as a template
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Single RNA polymerase (Prokaryotes) Core enzyme
Holoenzyme 2 ?,1 ? and 1 ? subunits 2 ?, 1
?, 1 ? subunits plus s subunit Polymerizes
RNA Finds initiation sites
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- 35 bases from initiation of transcription Recogn
ized by RNA polymerase
- 10 bases from initiation of transcription Unwind
ing of DNA double helix begins here
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Termination RNA polymerase recognizes signals
(sequence) for chain termination
Releases the RNA and enzyme from the template
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Simultaneous transcription and translation in
prokaryotes
Green arrow E. coli DNA Red arrow mRNA
combined with ribosomes
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Splicing removes the introns and brings together
the coding regions
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The Central Dogma(Francis Crick, 1958)
(Transcription)
(Translation) DNA ? RNA ?
Protein (Gene) (Phenotype) An
informational process between the genetic
material (genotype) and the protein (phenotype
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Translation (protein synthesis)
Peptidyl site peptidyltransferase attaches amino
acid to chain
Aminoacyl site new amino acid brought in
Ribosome moves in this direction
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Cells have adapter molecules called tRNA with a
three nucleotide sequence on one end (anticodon)
that is complementary to a codon of the genetic
code.

• There are different transfer RNAs (tRNAs) with
  anticodons that are complementary to the codons
  for each of the twenty amino acids.
• Each tRNA interacts with an enzyme
  (aminoacyl-tRNA synthetase) that specifically
  attaches the amino acid that corresponds to its
  anticodon.
• For example, the tRNA to the right with the
  anticodon AAG is complementary to the UUC codon
  in the genetic code (mRNA). That tRNA would
  carry the amino acid phenylalanine (see genetic
  code table) and only phenylalanine to the site of
  protein synthesis.
• When a tRNA has its specific amino acid attached
  it is said to be charged.

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Homopolymer was then added to a test tube
containing cell-free translation system, 1
radioactively labeled amino acid and 19 unlabeled
amino acids Proteins were isolated and checked
for radioactivity Procedure was repeated in 20
tubes, with each tube containing a different
radioactively labeled amino acid Only one tube
contained radioactively labeled protein the
amino acid that was labeled (phenylalanine) is
therefore specified by UUU
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Genetic Code

• Next synthesized heteropolymers
• The artificial RNA sequence would depend upon the
  ratio of the two or more NDPS added
• ADP and CDP in a 1 to 5 ratio
• 1/6 probability of incorporating an A being
  incorporated
• 5/6 probability of incorporating a C being
  incorporated
• The resulting RNA molecule would be a collection
  of different codons that are made-up of A and C
• The numbers of different codons in the RNA
  molecule is a matter of probability

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Genetic Code
ADP and CDP added in a 1 to 5 ratio AND if codon
is a triplet
Possible combinations Probability Possible codons Percent
3 A (1/6)3 0.4 AAA 0.4
2A 1C (1/6)2(5/6) 2.3 AAC, ACA, CAA 6.9 (2.3 2.3 2.3)
1A 2C (1/6)(5/6)2 11.6 ACC, CAC, CCA 34.8 (11.6 11.6 11.6)
3C (5/6)3 57.9 CCC 57.9
100
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Genetic Code
The poly (AC) RNAs produced proteins containing 6
amino acids
Amino acid Percent Possible codons
Proline 69 CCC (57.9)
Threonine 14
Histidine 12
Asparagine 2 2A 1C (2.3)
Glutamine 2 2A 1C (2.3)
Lysine 1 AAA (0.4)
2C1A (11.6)
1A 2C (11.6) 2A 1C (2.3)
1A 2C (11.6)
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Using the table below, can you translate this
nucleotide sequence? 5UUCGAUGCCCGGGGUCCUGAAAUUGU
UCUAGA 3

• The first step is to look for the AUG start
  codon.
• Next, group the nucleotides into a reading frame
  of 3 nucleotides per codons and use the table to
  find the amino acid that corresponds to each
  codon.
• Stop translating the mRNA when you reach a stop
  codon.
• Is this what you got?
• Met-Pro-Gly-Val-Leu-Lys-Leu-Phe-Stop

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Pathways
Gene A Gene B Enzyme A
Enzyme B Substrate
-------? intermediate ------? product
Most often the final product of the biochemical
pathway is something essential to life, like
amino acids, nucleotides, etc.
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Pathways
Gene A Mutant Gene B
Enzyme A No Enzyme B
Substrate -------? intermediate No Product
Can use mutants to work out pathways, and
identify which gene catalyzes which step
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growth - no growth
Minimal ornithine citrulline arginine No. s
Wild-type 4
Mut 1 - 3
Mut 2 - - 2
Mut 3 - - - 1
No. 3
No. 2
No. 1
arginine
citrulline
ornithine