from dna to protein: genotype to phenotype
DESCRIPTION
From DNA to Protein: Genotype to Phenotype. Biochemical Biosynthesis Pathways Lead to Understanding of Gene-Enzyme Relationship. Biosynthesis of Arginine. Ornithine transcarbamylase. Argininosuccinate lyase. Argininosuccinate synthetase. Acetylornithinase. N-acetylornithine. Ornithine. - PowerPoint PPT PresentationTRANSCRIPT
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12From DNA to Protein: Genotype to Phenotype
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Biochemical Biosynthesis Pathways Lead to Understanding of Gene-Enzyme Relationship
Biosynthesis of Arginine
Beadle & Tatum undertook the identification of mutations that blocked the synthesis of several vitamins & amino acids
Ornithinetranscarbamylase
Argininosuccinate synthetase
Argininosuccinate lyase
CitrullineOrnithineN-acetylornithine argininosuccinate arginine
aspartatecarbamyl phosphate
Acetylornithinase
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Mutagenesis Screen Identifies Link Between Genetic Element and Enzyme
Figure 14.24 Raven & Johnson, Biology 5th Ed
Mutagen
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Individual Mutants Blocked at Distinct Enzymatic Steps in Arg Biosynthesis Pathway
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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
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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
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DNA, RNA, and the Flow of Information• F. Crick coined phrase central dogma
DNA codes for RNA. RNA codes for protein.
• 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?
Transcription Translation
Replication
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Gene Expression: From Gene to Protein
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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)
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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)
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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
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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
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Gene Structure
• The DNA template Strand nomenclature
• For different genes in the same DNA molecule, the roles of the strands may be reversed
top, coding, sense5’
5’3’
3’
bottom, template, antisense
3’promoter coding region
5’5’3’coding strand
template strand
txn initiation site
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Transcription: DNA-Directed RNA Synthesis
• Initiation RNA polymerase binds to the promoter region
Coding
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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
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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
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Transcription – Prok v Euk
• Prokaryotic transcription occurs ___________
• Eukaryotic transcription occurs ____________
• Prokaryotic cells have ___________ RNA polymerase
• Eukaryotic cells have ____________ RNA polymerases
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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
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The Genetic Codebreakers
• 1960-65 M Nirenberg, G Khorana, P Leder
• Identified which nucleotide sequences specified which amino acids
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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
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The Genetic Code
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Anticodons & Wobble
RNA-RNA bp rules
A-U
G-C
G-U
Modified bases
Inosine (I)
I-A
I-U
I-C
5’-codon-3’ / 3’-αcodon-5’
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The Genetic Code
• How is the code read?
• 3letterwordsallruntogetherwhatspunctuation?
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Translation: Reading Frames
AAGCUAGCAUGUGGAUGCAUGAUCGCUACAAUCGAGGAUC
a: AAG CUA GCA UGU GGA UGC AUG AUC GCU ACA AUC GAG GAU C
b: A AGC UAG CAU GUG GAU GCA UGA UCG CUA CAA UCG AGG AUC
c: AA GCU AGC AUG UGG AUG CAU GAU CGC UAC AAU CGA GGA UC
Lys Leu Ala Cys Gly Cys Met Ile Ala Thr Ile Glu Asp
Ser stop His Val Asp Ala stop Ser Leu Gln Ser Arg Ile
Ala Ser Met Trp Met His Asp Arg Tyr Asn Arg Gly
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Putative Translation of cDNA Sequence
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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
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Translation - tRNA
• Intramolecular base pairing defines 2 structure
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Charging a tRNA Molecule
aminoacyl-tRNA synthetase
Amino acids attached to correct tRNAs by aminoacyl-tRNA synthetases
Phe-tRNAPhe
aa-tRNA
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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
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Ribosomes
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Electron Density Model of Ribosome & tRNAs
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Translation - Ribosome
A site – aa-tRNA binding site
P site - tRNA with peptide chain
E site – exit site empty tRNA briefly sits after translocation
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Translation Initiation
• Involves initiation factors to help ribosome & Met-tRNAi
Met bind
• Initiator tRNA enters P-site
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Translation Elongation
• Elongation factors aa-tRNA binding Translocation
• Peptidyl transferase Ribozyme activity of
large subunit
aa-tRNA entry
Peptidyl transferase
Translocation Termination
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Translation Elongation
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Polysomes
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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.
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Posttranslational Events
• Folding
• Glycosylation
• Phosphorylation
• Acylation
• Proteolytic processing
• Dimerization/multimerization
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Postranslational Events
• Subcellular location of translation and ultimate protein localization & modification
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Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 1)
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Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 2)
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AUGUGGCUCCCGAUUAA
Point Mutations
coding ATGTGGCTCCCGATTAAATGTGGCTCCTGATTAA
T C
AUGUGGCUCCUGAUUAA
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AUGUGGCUCCCGAUUAA
Point Mutations
ATGTGGCTCCCGATTAAATGTGGCTCCCGTTTAAcoding
AUGUGGCUCCCGUUUAA
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Point Mutations
ATGTGGCTCCCGATTAAATGTAGGCTCCCGATTAAcoding
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Point Mutations
ATGTGGCTCCCGATTAAATGTGGACTCCCGATTAAcoding
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Tautomeric Shifts Alter Base-Pairing Specificity
Keto-enol & amino-imino tautomerization
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Mutations Arise from Chemical Changes in Bases
Deamination
Alkylation