gene - sequence of bases in dna
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Gene - Sequence of Bases in DNA
5’ ATGCCTGCACATGTTAGC 3’ 3’ TACGGACGTGTACAATCG 5’
Specifies information about particular trait
Cellular phenotypes controlled by _ ?
GenerallyGene Protein Trait
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Evidence that genes code for enzymes
Garrod (1902) - ‘Inborn Errors of Metabolism’
Albinism - lack of pigmentation, melanin Lack: tyrosinase
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Evidence that genes code for enzymes
PKU (phenylketonuria) - accumulation of phenylpyruvic acid
Lack: phenylalanine hydroxylase
Mental retardation, seizures, fair skin, light sensitivity, musty odor
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Evidence that genes code for enzymes
Alkaptonuria - excrete homogentisic acid in urine (black)
Lack: homogentisic acid oxidase
Buildup of dark pigment in connective tissue
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Biochemical Pathways
Gene 1 Gene 2Gene 3
Enzyme 1 Enzyme 2Enzyme 3
A BC D
Blocked ifEnz 2 nonfunctional
Consequences???
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Tay-Sachs Disease
Symptoms: blind, deaf, unable to swallow, muscle atrophy, paralysisHigh incidence: East European and Ashkenazi Jews
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Beadle & Tatum - 1941 Neurospora crassa
Select auxotrophs thatdon’t grow on MEM
Determine AA required
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Determining Order of Intermediates
What is the order of intermediates?At which step is each mutant defective?
Precursor C A DB F
3 5 2 4 1
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Conclusions from Beadle & Tatum’s Work
One gene controls (encodes)
one protein or polypeptide subunit or functional RNA
(tRNA, rRNA, snRNA, miRNA)
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Sickle Cell Anemia
Defective Hemoglobin structure
Symptoms: fragile inflexible blood cells, anemia, blockage
heart failure, pneumonia, paralysis, kidney failure,abdominal pain, rheumatism
African American - 1 in 500 affected, 1 in 12 are carriersHispanic - 1 in 1,000 - 1,400 affected; Caucasian - rare
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Cystic Fibrosis
Defect: CF transmembrane conductance regulator chloride transport across membranes of some cells
Caucasians: Incidence - 1 in 2000; Carriers - 1 in 23Symptoms: pancreatic, pulmonary, digestive dysfunction
Life expectancy ~ 40 years
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How does a gene encode a protein?
DNA
5’ ATGCTAGTACTGATGCAGTCTGACTAC 3’
Polypeptide
amino - Phe - Arg - Pro - Lys - Thr - Ala - Cys - carboxyl
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Twenty common amino acids: protein subunits
Amino Acid Structure
H H OH - N - C - C - OH Ramino carboxylicgroup acid group
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Transfer of Information: Central Dogma
DNA RNAPolypeptide transcription translation
genes mRNA
rRNAtRNAsnRNAmiRNA
5’ CCT 3 ’ 5’ CCU 3’ Pro3’ GGA 5’
three bases (one codon) specify one amino acid
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Transcription: DNA - RNA
RNA polymerase
Promoter Initiation start site
Template Strand
RNA-like Strand (non-template)
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Gene Sequences Important in Transcription
Promoter - interacts with RNA polymerase, indicates start siteE. coli - consensus sequences
-35 (TTGACA) -10 (TATAAT)
Initiation Site of Coding Sequence -
Termination Sequences -
Upstream (-) Downstream (+)
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Initiation of Transcription (prokaryotes)
RNA polymeraseholoenzyme core enzyme -
2 , 1 , 1 ’
sigma factor - binds -35then -10
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Transcription Elongation and Termination
RNA polymerase - unwinds and rewinds DNA - proofreading
Terminator sequences Rho-dependent - protein involved in E. coli Rho-independent - RNA polymerase terminates
itself
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Transcription in Eukaryotes
RNA polymerases - ~ 12 subunitspol I - rRNA (28S, 18S, 5.8S) pol II - mRNA, snRNApol III - tRNA, 5S rRNA, snRNA
Promoter elements Core Inr - sequence spans +1
TATA box - at ~ -30 indicate start site Proximal CAAT box (~ -75)
GC box (~ -90)
enhance transcription
Enhancers - upstream or downstream of ORF
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Transcription Initiation in Eukaryotes
General transcription factors (GTFs) required to start
transcription
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Processing mRNA in Eukaryotes
3’ Poly A tail
transport
protection
Poly(A) site Poly(A) polymerase
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Processing mRNA in Eukaryotes
Intron removal by spliceosomes - snRNPs(small nuclear ribonucleoprotein particles)
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Translation
messenger RNA protein
Requirements:mature mRNA - instructions
charged tRNAs - bring amino acids
ribosome - workbench
initiation, elongation, termination factors
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Charging of tRNAAminoacyl tRNA synthetases - attach amino acid to 3’ end
Charged tRNA carries aa to ribosomeAnticodon binds complementary codon in mRNA
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Ribosomal RNA - rRNA
Mammalian ribosome
E. coli ribosome - 70S 50S - 23S rRNA, 5S rRNA, 34 proteins
30S - 16S rRNA, 20 proteins
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Ribosome Binding Site (RBS) for mRNA
Prokaryotes - 16 S rRNA binds mRNA - ~ 8-12 nucleotides upstream of start
consensus
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Ribosome Binding Site (RBS) for mRNA
Eukaryotes
Initiation factor eIF-4F binds 5’ cap
Other eIF proteins, 40S ribosome, initiator Met-tRNAmove along mRNA scanning for start codon
Start AUG embedded in Kozak sequence
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Initiator tRNAs
Prokaryotes - formylmethionine (fMet) + initiator tRNA
O H H OH - C - N - C - C - O - tRNA
RfMet - tRNA (fMet)
Eukaryotyes - special initiator tRNAs
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Deciphering the Genetic Code
Codons needed to specify 20 amino acids, 1 start, 3 stops = 24
Three letter codons would suffice.
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Deciphering the Genetic Code
Crick et al. 1961 - T4 phage - mutagenesis with proflavin
Frameshift mutations - downstream effect
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Deciphering the Genetic Code
Crick et al. 1961 - Experiments showed1 insertion suppresses 1
deletion1 deletion suppresses 1
insertion 3 insertions cause
suppression3 deletions cause
suppression
must be
triplet
code
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Deciphering the Genetic Code
Mixed polymers - mixtures of nucleotides synthesizedUsed for in vitro translation, protein product analyzed
Ex. 3/4 U + 1/4 G
RelativeCodon Probability Amount
AAUUU (3/4)3 = 27/64 1.0PheUGU, GUU, UUG 9/64 0.36Leu, Val, CysGGU, GUG, UGG 3/64 0.13Trp, Gly
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Deciphering the Genetic Code
Nirenberg + Leder - 1964
Ribosome-binding assays
Mix ribosomes with known codons in mRNA
Determine which tRNA (amino acid) binds
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Characteristics of the Genetic Code
Triplet codeContinuous5’CCGTATGACGCTACGTTAGACTTGACATC3’
NonoverlappingIncludes start and stop signals
Almost universal (mammalian mitochondria, Tetrahymena)
DegenerateWobble occurs
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