Gene - Sequence of Bases in DNA

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1 Gene - Sequence of Bases in DNA 5’ ATGCCTGCACATGTTAGC 3’ 3’ TACGGACGTGTACAATCG 5’ Specifies information about particular trait Cellular phenotypes controlled by _ ? Generally Gene Protein Trait

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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 _ ?. Generally Gene Protein Trait. 1. Evidence that genes code for enzymes . - PowerPoint PPT Presentation

Transcript of 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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Evidence that genes code for enzymes

Phenylalaninehydroxylase

Tyrosinase

Homogentisicacid oxidase

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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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Human Genetic Diseases - Table 4.2

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Beadle & Tatum - 1941 Neurospora crassa

Select auxotrophs thatdon’t grow on MEM

Determine AA required

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One-Gene-One-Enzyme Hypothesis

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Genetic Analysis of Biochemical Pathways

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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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Mutations Responsible for Sickle Cell Anemia

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Examples of Hemoglobin Mutations

Hb-C - mild anemia

Many changes have only slight effects

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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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Protein Structure

Primary Structure

Secondary

Tertiary

Quaternary

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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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RNA Polymerase ActivityUnwinding & Synthesis

5’ nucleotidetriphosphate

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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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Products of Transcription

RNAprocessinginEukaryotes

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Processing mRNA in Eukaryotes

5’ capping

Nuclease protection

Ribosome binding

7-methylguanosine

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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

RNA

Splicing

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Processing mRNA in Eukaryotes

Intron removal by spliceosomes - snRNPs(small nuclear ribonucleoprotein particles)

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Processing mRNA in Eukaryotes

Self- Splicing Introns

Ribozymes

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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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Transfer RNAs - products of several genes

tRNA

Anticodon3’ end

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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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tRNA Landing Sites

E (exit) P (peptide)A (aa)

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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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Initiation of Translation

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Elongation during Translation

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Elongation during Translation

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Peptidyl transferase

A site3

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Peptide Bond Formation

Peptidyl transferase

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Elongation during Translation

3

4

translocation 5’ toward 3’

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Elongation during Translation

next tRNA binds

Elongation continues until stop codon

5

6

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Termination of TranslationStop codons: UAG, UAA, UGA

Release or Termination Factors (RF)

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Overview of Translation

Colinearity of mRNA codons and amino acids in polypeptide

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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

Intragenic suppression

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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

Nirenberg + Khorana - 1968 Nobel PrizeSynthetic mRNAs

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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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Genetic Code Table (mRNA)

Degenerate code

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Why is the genetic code degenerate?

wobble

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Wobble Rules