2.6/2.7 DNA Flashcards
(27 cards)
Explain DNA structure
2 antiparallel polynucleotide strands
linked by H bonds bet. complementary base pairs
Phosphodiester bonds between adjacent nucleotides
2 chains coil around each other to form right handed double helix
Watson and Crick did what
Introduced double-helical model for the structure of DNA
- 2 polynucleotide strands
- nitrogenous bases arranged on the inside
- Parallel strands held by H bonds
- 10 bp per turn of helix
DNA vs RNA
length - long strands, millions of nucleotides vs short strands, several thousand
sugar - deoxyribose vs ribose
bases - T vs U
form - double strand, double helix vs mRNA, tRNA, rRNA, single strnads
Meselson and Stahl did what, also explain their experiment
Explain why other models were hence proven wrong
Semiconservative model - replication of DNA - each daughter molecule will have one old strand and one newly made strand
- Grow bacteria in 15N media
- Transfer to 14N media and grow for <1, 1.0, 2.0, 3.0 generations
- Isolate DNA after each generation, transfer to CsCl gradient, centrifuge.
- Density gradient formed, most Cs and hence greatest density at the bottom.
- Substance centrifuged with CsCl becomes concentrated at the level corresponding with its density
- DNA with heavy nitrogen end near base, light nitrogen end near top
- Observe stained DNA under UV
First rep produced band of hybrid DNA - not conservative
Second replication produced both light and hybrid - eliminating dispersive model
PCR process
Copy a small sample of DNA many times
Repeated cycling through high and lower temperatures to promote melting and annealing of DNA strands
- Heated to high temps to break H bonds between strands, separation in process of denaturing
- Excess primers added to ensure strands do not anneal back tgt, bind to targeted DNA sequences at lower temp through process of annealing
- Taq polymerase added, can withstand high temperatures. Forms new double stranded DNA by adding complementary bases through process of elongation.
Rosalind and WIlkins did what
Determined double helix structure, number of nucleotides per turn
Produced xray diffraction patterns by bombarding crystalline DNA with Xrays, measuring intensities and scattering angles of X rays leaving the material.
Hershey and chase experiment T2 bact
Engineered 2 batches of T2 bacteriophage - one with radioactive phosphorous in DNA and one with radioactive sulfur in protein.
Specific as protein does not contain phosphorous, DNA does not contain sulfur.
2 identical sets of ecoli exposed to diff T2
Agitation to free outside phage parts from cell, centrifuge to form pellet. radioactivity was found in supernatant for S, found in pellet for P, showing DNA enters the bacteria
Nucleosome def
Consists of 8 histone molecules to form a bead, DNA double helix is looped around bead twice. H1 histone binds DNA molecule to bead.
Nucleosome regulates transcription by controlling access to selected lengths of DNA.
Non coding regions of DNA examples and functions (6)
Regulators of gene expression: Enhancers and silences
Introns: Separate exons in eukaryotic genes, alternative splicing
Telomeres: Multiple repetitions of short nucleotide sequence, occur at ends of DNA molecules, prevent gene erosion
tRNA genes: Code for short lengths of RNA used to transfer amino acids from cytoplasm to growing polypeptide chain in ribosome
Promoters: binding site of RNA polymerase during transcription
VNTR: Lengths of noncoding DNA, repeated numerous times, found in clusters
Leading v lagging strand
Continuous vs okazaki fragments
Extends in direction of replication fork vs extends in opposite direction
Requires only 1 primer, requires more than one primer
DNA replication - essay
- Nucleotide added initially deoxyribose triphosphate, but after addition pyrophosphate is removed. Hydrolysis of pyrophosphate provides energy for replication
- Replication begins at origin of replication
- Semiconservative
- Helicase, unwinds DNA at replication fork, break H bonds, 2 template strands
- Topoisomerase release strain
- Single stranded binding proteins keep strands apart
- Primase adds RNA primer, complementary base pairing
- Polymerase III binds adjacent to primer on template strand, initiates replication, links nucleotides, phosphodiester bonds, 5’-3’ direction, complementary base pairing.
- Towards rep fork on leading, away on lagging
- forms okazaki fragments on lagging, primase has to synthesize primer at 5’ end of each fragment
- DNA polymerase I replace primer with DNA, but nick present
- Ligase seals nick between okazaki fragments, between rest of strand and DNA molecule that replaces primer.
- Proof reading by pol. I and III
Sanger dideoxy sequencing
- dideoxy chain termination method
- modified nucleotides: dideoxyribonucleotides (ddNTP) tagged with fluorescent label
- Replication stopped when ddNTP is added to growing chain. Nucleotide that ends each strand identified with fluorescent label.
- Create many copies of DNA in presence of ddNTPs, excess or regular nucleotides.
- Separate copies with AGE, read fluorsecent tag on ddNTP to read order of nucleotides.
- Read DNA sequence from resulting spectrogram.
DNA profiling process
Obtain sample
PCR to amplify copies and tandem repeats
AGE to separate DNA into bands according to number of repeats
Pattern of bands is unique to individual
Synonyms of coding strand
Synonyms of template strand
Sense = coding = nontemplate = codon
Antisense = noncoding = template = anticodon (ANT)
Transcription - essay
- Definition - synthesis of mRNA using information in DNA
- Antisense acts as a template is transcribed
- Polymerase attach
- Unwind, seperate, break h bonds
- expose 10-20 base pairs for rna nucleotides to bind
- binding, complementary base pairing, AUGC, 5-3
- Formation of h bonds bet complementary base and rna
- rna nucleoside triphosphate used, 2 phosphate groups removed for energy
- terminator reigion
- transcription stops, rna polymerase and mrna released
- reform double helix
Post transcriptional modification explain
Alternative splicing explain
RNA splicing - removal of introns, exons left behind by spliceosome to form mature mRNA
Alternative splicing - the same gene can encode more than 1 polypeptide, depending on which segments are treated as exons during splicing. -more proteins than genes.
Addition of 5’ cap = add modified G molecules at 5’ end, help ribosomes attach for translation
Poly A tail - addition of A molecules to 3’ end
- increase stability
- facilitates export of mrna to cytoplsam
- prevents degradation by hydrolytic enzymes
Epigenetic inheritance definition
Inheritance of traits not transmitted by mechanisms involving nucleotide sequences
Promoter definition
Nucleotide sequence in DNA, binding site for mRNA polymerase, initiates transcription but is not transcribed
Regulation of transcription
Genomic imprinting def
Chemical modification of histones
- double helix looped around histones to form nucleosomes
- End of histones project outwards from nucleosome, inhibit or facilitate transcription.
- Addition of acetyl groups to positively charged lysine loosens tight binding, allow access to transcription enzymes and proteins.
- Addition of methyl groups to AA condense chromatin, inhibiting transcription. Phosphorylation next to methylated AA loosesns chromatin, promotes transcription.
DNA methylation
- addition of methyl groups to DNA bases - long term inactivation of genes in cellular differentiation. Silencing of gene expression.
- genomic imprinting - methylation regulates expression of maternal/paternal alleles of certain genes at start of development
Enhancers, silencers, transcription factors
- Before transcription, RNA polymerase binds to transcription factors, transcription complex, promoter region before start of gene
- Transcription factor = proteins regulate rate of transcription
Enhancer/silencer = regulatory sequence on DNA, increase/decrease rate
Promoter proximal elements = sequence near to promoter, binding with proteins affects transcription
Distal control elements = enhancer/silencer located far away from gene/in intron.
Environmental factors affect gene expression (see later)
Prokaryotic transcription regulation explain
Lactase production in ecoli example
- Regulation of transcription by responding to environmental change
Eg. regulation of enzyme production by feedback inhibition
- repressor protein for lactase gene deactivated in presence of lactose
- RNA polymerase can bind to template strand, transcription, express gene, lactase produce, lactose broken down
- no more lactose, repressor no longer deactivated, attaches to operator, blocks transcription of lactase gene.
Feedback inhibition, repressor protein, lactase gene, deactivated, operator, transcription
WObble?
Flexible pairing at the third base of codon results in redundancy, less chance mutation will result in change in AA produced
Describe structure of eukaryotic ribosome
- 2 subunits, 1 large, 1 small
- 3 binding sites for tRNA on large subunit (aminoacyl, peptidyl, exit)
- 2 tRNA molecules bind at same time on ribosome
- 1binding site for mRNA on small subunit
- rRNA catalyzes formation of peptide bond
A, P, E sites significance
A - aminoacyl: anticodon of incoming tRNA-AA binds with specific mRNA codon via cbp with tRNA anticodon. Holds tRNA that carries next AA to be added to the chain
P - peptidyl: AA carries the growing polypeptide chain, forms peptide bond with subsequent AA to be added
E - exit: tRNA exits the ribosome after transferring AA to growing polp. chain.
Translation essay
Define translation - conversion of base sequence on mRNA into amino acid sequence by initiation, elongation, termination
tRNA:
- aminoacyl-tRNA synthetase link tRNA to AA
- tRNA each have specific anticodon and carries specific AA
mRNA and ribosome:
- mRNA bind to small subunit
- Small and large unit assemble
- mRNA carries codons, one codon = 1 AA
- anticodon on tRNA pairs with codon on mRNA via cbp.
Transcription:
- ribosome slides to start codon
- Anticodon for methionine pairs with start codon (AUG)
- first tRNA binds to P site, next codon binds to A site.
- Peptide bond forms between AAs on A and P site
- Ribosome moves to next codon in 5’ to 3’
- tRNA shifts from A to P, P to E
- First tRNA that has lost its AA released from ribosome at E
- Another tRNA enters A site, binds to next codon.
- continues to elongate until stop codon reached
- stop codon causes release of polypeptide from ribosome.
