From DNA to Protein:
Genotype to Phenotype
Biochemical Biosynthesis Pathways Lead to Understanding of Gene-Enzyme
Relationship
Biosynthesis of Arginine
Acetylornithinase
Ornithine
transcarbamylase
N-acetylornithine
Ornithine
Argininosuccinate
synthetase
Citrulline
carbamyl
phosphate
Argininosuccinate
lyase
argininosuccinate
aspartate
Beadle & Tatum undertook the identification of mutations that
blocked the synthesis of several vitamins & amino acids
arginine
Mutagenesis Screen Identifies Link Between Genetic Element and Enzyme
Mutagen
Figure 14.24
Raven & Johnson, Biology 5th Ed
Individual Mutants Blocked at Distinct Enzymatic Steps in Arg Biosynthesis Pathway
What is a Gene
• A gene is a contiguous region of DNA that is transcribed
• The transcript (that which is transcribed) is an RNA molecule
• There are 3 types of genes & 3 types of RNA transcribed
 rRNA encoding genes  rRNA (class I)
 protein encoding genes  mRNA (class II)
 tRNA encoding genes  tRNA (class III)
• In eukaryotic cells, each class of RNA is transcribed by a
different RNA polymerase
Decoding The Coding Problem
• 1959-60 – F Crick, S Brenner, F Jacob, M Meselson, J
Monod
 Messenger Hypothesis
 RNA serves as intermediate btwn DNA & protein synthesis
 Ribosomes associated with protein synthesis
 Heterogeneous RNA (hnRNA) found w/ & w/o ribosomes
 Is hnRNA or rRNA the messenger?
 Brenner, Jacob & Meselson did a 1 week experiment that
proved hnRNA was the message – renamed mRNA
DNA, RNA, and the Flow of Information
• F. Crick coined phrase central dogma
 DNA codes for RNA. RNA codes for protein.
Replication
Transcription
Translation
• How is expression of gene controlled?
• How does information get from the nucleus to the cytoplasm?
• What is relationship btwn DNA nucleotide sequence & protein
amino acid sequence?
Gene Expression: From Gene to Protein
Decoding The Coding Problem
• Crick proposed the Adaptor Hypothesis
 intermediate btwn mRNA & protein synthesis
 intermediate adapt (bind) to mRNA & “decode” the
message
• What is nature of genetic code?
 1 to 1
 2 to 1
 3 to 1
 4 to 1
• Nature of the adaptor?
 tRNA –necessary for translation
 tRNAs w/ amino acids attached
 Aminoacylated tRNA (aa tRNA)
DNA, RNA, and the Flow of Information: The Central Dogma
• Gene expression
 The production of an ultimate gene product (RNA &/or
protein)
• The expression of a gene takes place in two steps:
 Transcription – production of a single-stranded RNA copy
of a segment of DNA
 Translation – production of a protein from mRNA
• Gene product is therefore
 rRNA or tRNA or protein (via mRNA)
Review of RNA
• RNA differs from DNA
 single stranded
 ribose
 uracil
• RNA can exist in a double-stranded complex with either DNA, with itself,
or with another RNA strand
• mRNA – encodes proteins
• rRNA – main constituent of ribosomes
• tRNA – transfer amino acids to ribosome and decode mRNA
• snRNA – splicing
• snoRNA – RNA modifications
• 7SL RNA – co-tranlational translocation for secretion
• siRNA – regulation of transcription & translation
Transcription: DNA-Directed RNA Synthesis
• Requirements:
 A DNA template
 ribonucleoside triphosphates (ATP, GTP, CTP, and UTP)
 RNA polymerase
• Regulated process
 transcription factors
 DNA sequences recognized by RNA pol & txn facs
Gene Structure
• The DNA template
 Strand nomenclature
5’
top, coding, sense
3’
3’
5’
bottom, template, antisense
• For different genes in the same DNA molecule, the roles of
the strands may be reversed
txn initiation site
5’
3’
promoter
coding strand
template strand
coding region
3’
5’
Transcription: DNA-Directed RNA Synthesis
• Initiation
 RNA polymerase binds to the promoter region
Coding
Transcription: DNA-Directed RNA Synthesis
• Elongation
 RNA polymerase unwinds the DNA and synthesizes RNA
 Nucleotides added at 3’ end of growing RNA strand
 5  3
 Template and RNA transcript are antiparallel
Transcription: DNA-Directed RNA Synthesis
• Termination
 RNA polymerase reaches DNA sequences at end of gene
that cause it to stop and release the RNA and DNA
Transcription – Prok v Euk
• Prokaryotic transcription occurs ___________
• Eukaryotic transcription occurs ____________
• Prokaryotic cells have ___________ RNA polymerase
• Eukaryotic cells have ____________ RNA polymerases
The Genetic Code
• The genetic code relates nucleotide sequence of genes
(DNA/mRNA) to the amino acid sequence of proteins
• What is the nucleotide-amino acid correspondence?
 A degenerate code
 Frames
• How many nucleotides correspond to an amino acid?
 Three
 A triplet code
The Genetic Codebreakers
• 1960-65 M Nirenberg, G Khorana, P Leder
• Identified which nucleotide sequences specified which amino
acids
The Genetic Code
• A codon was determined to be 3 adjacent nucleotides
• Code is degenerate
 Multiple codons specify same amino acid
 Each of these codons is NOT recognized by a different
tRNA
 “wobble” in the base-pairing btwn tRNA anticodon w/
mRNA codon
The Genetic Code
Anticodons & Wobble
5’-codon-3’ / 3’-αcodon-5’
RNA-RNA bp rules
A-U
G-C
G-U
Modified bases
Inosine (I)
I-A
I-U
I-C
The Genetic Code
• How is the code read?
• 3letterwordsallruntogetherwhatspunctuation?
Translation: Reading Frames
AAGCUAGCAUGUGGAUGCAUGAUCGCUACAAUCGAGGAUC
a:
AAG CUA GCA UGU GGA UGC AUG AUC GCU ACA AUC GAG GAU C
Lys Leu Ala Cys Gly Cys Met Ile Ala Thr Ile Glu Asp
b:
A AGC UAG CAU GUG GAU GCA UGA UCG CUA CAA UCG AGG AUC
Ser stop His Val Asp Ala stop Ser Leu Gln Ser Arg Ile
c:
AA GCU AGC AUG UGG AUG CAU GAU CGC UAC AAU CGA GGA UC
Ala Ser Met Trp Met His Asp Arg Tyr Asn Arg Gly
Putative Translation of cDNA Sequence
Translation - tRNA
• tRNA has three functions:
 carries amino acid
 base-pairs with mRNA
 interacts with ribosomes
• tRNAs must read mRNA correctly to assure proper protein
sequence
• tRNAs must carry the correct amino acids
Translation - tRNA
• Intramolecular base pairing defines 2 structure
Charging a tRNA Molecule
Amino acids attached to correct tRNAs by aminoacyl-tRNA synthetases
aminoacyl-tRNA
synthetase
aa-tRNA
Phe-tRNAPhe
Translation – Ribosomes
• 2 subunits: Large & Small
• Eukaryotes
 Large – 60S – 28S, 5.8S & 5S rRNA + ~45 proteins
 Small – 40S – 18S rRNA + ~ 33 proteins
 Ribosome – 80S
• Prokaryotic
 Large – 50S – 23S & 5S rRNA + ~ 40 proteins
 Small – 30S – 16S rRNA + ~ 28 proteins
 Ribosome – 70S
Ribosomes
Electron Density Model of Ribosome & tRNAs
Translation - Ribosome
 A site – aa-tRNA binding site
 P site - tRNA with peptide chain
 E site – exit site empty tRNA briefly sits after translocation
Translation Initiation
• Involves initiation factors to
help ribosome & Met-tRNAiMet
bind
• Initiator tRNA enters P-site
Translation Elongation
aa-tRNA entry
Peptidyl
transferase
Translocation
• Elongation factors
 aa-tRNA binding
 Translocation
• Peptidyl transferase
 Ribozyme activity of
large subunit
Termination
Translation Elongation
Polysomes
Regulation of Translation
• Antibiotics
 defensive molecules produced by fungi & bacteria, against
other microbes
• Molecular modality
 synthesis of cell walls, inhibiting transcription, inhibiting
translation
 erythromycin
 streptomycin
 tetracycline
• Because of differences between prokaryotic and eukaryotic
ribosomes, the human ribosomes are usually unaffected.
Posttranslational Events
• Folding
• Glycosylation
• Phosphorylation
• Acylation
• Proteolytic processing
• Dimerization/multimerization
Postranslational Events
• Subcellular location of translation and ultimate protein
localization & modification
Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 1)
Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 2)
Point Mutations
T
coding
C
ATGTGGCTCCCGATTAA
ATGTGGCTCCTGATTAA
AUGUGGCUCCUGAUUAA
AUGUGGCUCCCGAUUAA
Point Mutations
coding
ATGTGGCTCCCGATTAA
ATGTGGCTCCCGTTTAA
AUGUGGCUCCCGAUUAA
AUGUGGCUCCCGUUUAA
Point Mutations
coding
ATGTGGCTCCCGATTAA
ATGTAGGCTCCCGATTAA
Point Mutations
coding
ATGTGGACTCCCGATTAA
ATGTGGCTCCCGATTAA
Tautomeric Shifts Alter Base-Pairing Specificity
Keto-enol & amino-imino tautomerization
Mutations Arise from Chemical Changes in Bases
Deamination
Alkylation