Genetics Exam 1

DNA types

chromosomal DNA in the nucleus

mitochondrial DNA

chloroplast DNA

bacterial DNA (chromosomes and plasmids)

viral DNA

RNA types

messenger RNA

ribosomal RNA

transfer RNA

ribozymes--act as enzymes

antisense RNAs-regulate gene activity (act in a gene-specific manner)

small nuclear RNAs help process pre-mRNAs into mRNAs

Transformation Principle

* Discovered by Fred Griffith
* Used Virulent and Non Virulent Pneumonia
* Taught Us That One Bacterium Can Transmit Its Characteristics To Another

DNA is transforming principle

* Avery, MacLeod and McCarty
* They performed an experiment similar to Griffith, but they treated the virulent form with 3 different enzymes: Protease, Rnase, Dnase
* Dnase -→ destroyed DNA -→ animal lived

Hershey-Chase Experiments

* Used Radioactive sulfur-to trace protein.
* Radioactive phosphorus- to trace DNA
* Radioactive phosphorus transferred and sulfur did not
* further confirmed DNA as the transformation factor

Purines

* A(30%)
* G(20%)
* Two rings

Pyramidines

* T(30%)
* C(20%)
* Single ring

Rosalind Franklin and Maurice Wilkins

* DNA is helical and made of 2 \n parallel parts
* The helix contains 10 nucleotides \n (nt) per turn
* The helix has a diameter of 20 \n angstroms (2 nm)

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James Watson and Francis Crick

* DNA is double stranded and \n runs antiparallel.
* Bonds between A-T and G-C \n hold the 2 strands together.
* Complementary basing \n suggests a mechanism for \n replication

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Primary Structure of DNA

* Phosphate + Pentose Sugar + Nitrogenous Base

DNA strand directionality

* 5’ Carbon of one sugar is linked to the phosphate group
* The phosphate group is then linked to the 3’ C of the next nucleotide

DNA configurations

B form -→ typical configuration in vivo, right handed

A form -→ under high salt configurations, more compact, right handed

Z form -→ seen in short DNA pieces with alternating purine/pyrimidine bases, left handed

Conservative Model of DNA replication

* The original double strand serves as a template for a new molecule of DNA

Dispersive Model of DNA replication

* Both DNA molecules break down into fragments and serve as a template for the new fragment

Semi-conservative Model of DNA replication

* the original strand unwinds and is used as a template to generate the new strand

Meselson and Stahl’s Experiment

* Used 14N and 15N
* If dispersive -→ all DNA would be intermediate weight -- every strand has mixture of old and new DNA
* Because some DNA was light and some was intermediate -→ semi conservative

Theta Replication

1. Double strand unwinds-- creating replication bubble to be used as templates
2. Unwinding continues -- bubble gets larger (bidirectional)
3. DNA synthesis proceeds in the 5’→ 3’ direction

Rolling Circle Replication

1. Single strand break creating a 3’ OH and 5’ P group
2. New nucleotides are added on the 3’ OH break using the inner strand as a template
3. As new strand gets elongated, original strand gets displaced
4. Linear fragment that rolls off can be used as a template for a new strand

Unidirectional

Linear Replication

* Multiple Origins


1. At each origin DNA unwinds to create a replication bubble
2. DNA synthesis takes place on both strands, replication forks proceed outwards
3. Forks eventually run into each other and DNA segments fuse -→ producing two identical linear DNA molecules

Bidirectional

Stages of Prokaryotic DNA Replication: 1. Initiation

DnaA protein binds to the origin of replication (oriC)

* Opens a small site for helices and single strand binding protein to enter and begin unwinding the two strands

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Stages of Prokaryotic DNA Replication: 2. Unwinding

DNA replication requires a single strand template -- multiple enzymes needed -- DNA Helicase, Single Strand Binding Protein, DNA Gyrase

DNA Helicase (unwinding)

Breaks the Hydrogen bonds between the bases

Single Strand Binding Protein (unwinding)

Bind to the single strands once they form to stabilize the structure

DNA Gyrase (unwinding)

Topoisomerase

* Prevents supercoiling and upstream torsional strain the DNA builds up during unwinding

Topoisomerase I(unwinding)

Creates single stranded breaks in DNA

Topoisomerase II (unwinding)

* DNA Gyrase
* breaking the double strand, passing the double helix through it and resealing it again

Stages of Prokaryotic DNA Replication: 3. Elongation

* Single strands of DNA are used as template to make new strand
* Poly I removes the RNA primer and replaces it with DNA
* single break remains between the DNA generated by Poly I (replacing the RNA primer) and poly III, break sealed by ligase

Primers (elongation)

* Primase is used to generate a short 10bps of RNA which ends in a free 3’ OH group, RNA is late removed and replaced with DNA

DNA polymerase II (elongation)

* replications DNA using the RNA 3’-OH group
* Works on leading strand
* 5’ -→ 3’ activity for replication
* 3’ → 5’ exonuclease activity for backing up and replacing errors

DNA Polymerase I (elongation)

* removes the RNA primer and replaces it with DNA
* Works on lagging strand
* 5’→ 3’ activity for replication
* 5’ → 3’ exonuclease activity to remove primers and replace with DNA
* 3’→5’ exonuclease activity for backing up and replacing errors

Stages of Prokaryotic DNA Replication: 4. Termination

* Some DNA replication terminate when the two replication forks meets
* others need assistance of Tus
* Tus binds to Ter sequence → blocks replication

Differences between Prokaryotic and Eukaryotic DNA replication: Origin

Prokaryotes → one origin of replication

Eukaryotes → multiple origins

Differences between Prokaryotic and Eukaryotic DNA replication: Cell Cycle

Prokaryotic → always replication

Eukaryotic → controlled by cell cycle

Differences between Prokaryotic and Eukaryotic DNA replication: Licensing

1. Origins are licensed → Licensing portions (origin recognition complex - ORC) get attached to origin
2. Initiation machinery (MCM2-7) binds and starts replication. Licensing factor is removed as replication fork moves away from origin → removed to ensure that replication can occur more than once per cycle

Eukaryote DNA Polymerase: Alpha

* has primase activity
* Initiates DNA synthesis by generating a short primer

Eukaryote DNA Polymerase: Delta

* takes over after the primer has been laid down on the lagging strand

Eukaryote DNA Polymerase: Epsilon

* Functions like delta but works on leading strand

Differences between Prokaryotic and Eukaryotic DNA replication: Nucleosomes

* eukaryotic DNA packaged for stability with histones
* Nucleosome has 8 histones
* DNA needs to unwrap and histones need to be removes so DNA can be replicated


1. Original nucleosome removed
2. Some of the old histone find their way to their location on the new strand
3. new histones are made and attach to the old histones

Telomeres

* repeat DNA sequence at the end of each chromosome
* telomere of lagging strand cannot be replicated
* Human telomeres have a repeated sequence: TTAAGGG → repeated up to 2500 times in newly created cell’s telomeres
* do not code for any protein, meant to be a buffer so as DNA gets shorter, critical genes are not impacted

Telomerase

* can keep telomere from shortening
* RNA portion of telomerase binds to DNA and acts as template for new synthesis
* protein portion of telomerase synthesizes new DNA to fill the telomere
* no exposed 3’ end → unknown how synthesis occurs

Process whereby Genes make Proteins Transcription and Translation RNA processing

Transcription (Dan makes pre mRNA) → Post-Transcriptional RNA Processing (Introns spliced out of pre-mRNA, 5’ end capped w a methyl guanine, 3’ end gets poly-A tail) → Translation ( ribosome reads mRNS, strings amino acids together) → Post Translation Processing (folding, adornment with chemical side groups, localization, possibly enzymatic cleavage, possibly joining with other proteins to form heteromers)

Transcription

* production of RNA from DNA template
* one strand copied, other strand -- non template strand

Transcription Unit

* promoter, the RNA coding sequence, and the terminator
* copied: everything but promoter

Bacterial Transcription: Initiation

* assembly of transcription machinery
* transcription machinery needs to find promoter
* consensus sequence

Bacterial Transcription: Elongation

* DNA is made into RNA using RNA polymerase

Bacterial Transcription: Termination

* Separation of the RNA molecule from the parent DNA strand

Rho-Dependent terminators (bacterial transcription termination)

1. RNA polymerase stops at terminator DNA
2. Rho attaches to a sequence on the RNA strand called the rho utilization site (rut)
3. Rho moves to the 3’ end unwinds the RNA from the DNA bring transcription to an end

* based on an inverted repeat sequence that causes formation of secondary structure
* destabilizes structure

Eukaryotic Initiation: Nucleosomes

* histones grip DNA so tightly that transcription factors cannot get in to the bind w promoter
* acetylation of lysines in the histone proteins loosens the grip the histones have DNA -- lysine is positively charges so it tends to bind the negatively charged DNA tightly
* adding negatively charged acetyl groups on the lysine loosens the histones’ hold on the DNA
* allows transcription factors to bind to promoter region

RNA Polymerase II (Eukaryotic Initiation)

* assembles on the promoter with another 50 polypeptides -- creates open bubble for transcription to begin

Eukaryotic Elongation

* about 30bps are generated before polymerase enters this phase
* initiation factors will remain on the promoter if the protein is needed at high levels

Eukaryotic Termination

* depends on polymerase used
* poly I → uses a protein that binds to the downstream DNA sequence
* Poly II → can continue to work for 100s of nucleotides past its stop, stopped by Rat1 or Xrn1
* Poly III → transcribes a long stretch of uracils

Prokaryotic Gene Organization

* colinear
* direct relationship between # of nucleotides and amino acids

Eukaryotic Gene Organization

* DNA nucleotides that do no transcribe into protein
* RNA in the nucleus looks different than in the cytoplasm
* genes contain exons(coding) and introns(non-coding)
* direct relationship between intron size and organism complexity// more complex → longer in introns
* introns are spliced out as part of RNA processing procedure

Shine Delgarno Sequence

* In prokaryotic RNA → stabilizes ribosome

Eukaryotic RNA processing: 5’ Cap

* methylated guanine cap is put on 5’ end of mRNA
* usually 3 phosphate groups on 5’ end of nucleotide 1
* one is removed -- a 5’-5’ triphosphate linkage is made

Eukaryotic RNA processing: Polyadenylation

* poly A tail is added at 3’ end
* requires polyadenylation signal -- consensus sequence
* stabilizes RNA

Eukaryotic RNA processing: Splicing

* help form spliceosome which is responsible for removal intron
* also tells spliceosome where to cut
* alternative splicing allows one gene to make several different isoforms of its protein

RNA can be edited after transcription

* guide RNAs bind to the mRNA and cause it to be edited
* nucleotides can be added or deleted, and one nucleotide can be substituted for by another
* resultant mRNA base sequence/ protein’s amino acid sequence will not match those predicted by the DNA sequence

Genetic Code

* determines the nucleotide sequence
* triplet code-- three nucleotides from the mRNA encode a single amino acids
* 4 different nucleotides, 3 locations in the triplet code
* 64 possible codons for 20 different amino acids

Genetic Code: Degeneracy

* 3/64 are stop codons
* 61 sense codons
* a single amino acid may be specified by more than one codon

tRNAs carry amino acids in for translation

* mRNA codon sequence dictates which anticodon the tRNA must have
* each tRNA with a given anticodon sequence always carries the same amino acid
* maintains the fidelity of the genetic code
* 20 different aminoacyl-tRNA syntheses
* each one attaches a different amino acid to the appropriate tRNAs

Wobble may exist in the pairing of codon and anticodon

* 1st and 2nd base of the codon base
* hydrogen bonds between them making the 3rd codon require less energy to come together. There is more flexibility here

Reading Frame

* is critical to establish before translation begins
* established by the start codon
* only allows for 1 reading frame

Initiation of translation in bacteria

* small subunit of the bacterial ribosome attaches to the shine-delgarno sequence in the bacterial mRNAs

Initiation factors

* help ribosome assemble


1. small subunit lands on the 5’ UTR (consensus sequence) and finds start codon
2. Start tRNA attaches to the mRNA
3. Large subunit binds to the complex

Shine Delgarno Sequence

Prokaryotic mRNA consensus sequence

* in Eukaryotes → 5’ cap and 3’ poly A tail bond bind to initiation factors that stabilize the structure

Kozak Sequence

* Eukaryotic consensus sequence around the START codon

Elongation of polypeptides during translation

1. Incoming aminoacyl tRNA - new tRNA moves into A site, where anticodon base pairs with the mRNA codon
2. Peptide bond formation - the amino acid attached to the tRNA in the P site is transferred to the tRN in the site
3. Translocation - Ribosome moves down mRNA -- tRNA attached to the polypeptide chain moves into P site -- site is empty

Termination of translation

* ribosome needs to reach a stop codon -- release factor binds to ribosome at that point
* RF bind to A site → polypeptide is release from P site -- GTP is hydrolyzed → disassociation of ribosome from mRNA

Nonsense-mediated mRNA Decay

* mutation that creates a STOP codon at site of mutation
* polypeptide is not completed
* handles premature STOPs
* normal transcript = exon-exon - junction proteins removed
* premature stop = exon-exon - proteins remain → mRNA decay

Unstopped mRNAs must also be degraded

* mutation changes the STOP codon to an amino acid codon → translation is complete and there’s no STOP codon in the mRNA → causes ribosome to get stuck on that mRNA, and leaves it unavailable to perform more translation → signals the degradation of the mRNA

Nonstop mRNA Decay

* if ribosome gets to the end of the mRNA-- has not encountered a STOP codon -- ribosome’s A site will be hanging off the edge of the mRNA -- nothing to fill the ribosome’s A sit
* signals proteins that attach to the mRNS and degrade from its 3’ end forward

Proteins must be folded into their 3D shapes

* hydrophilic amino acids are typically found on the surface of the protein
* hydrophobic amino acids are typically found buried inside the 3D protein structure

Levels where gene expression can be regulated

DNA (Transcriptional (Cis/trans genetic regulation// Epigenetic regulation) )→ RNA (Post-transcriptional regulation (Processing and stability//miRNA // sequestration) → Protein (Post-Translational Regulation (Reversible: Modification // Irreversible: Degradation))

Positive Control

* Increase expression
* Regulatory protein → activator → stimulates transcription

Negative control

* decrease expression
* Regulatory protein → repressor → inhibits transcription

DNA Binding Domain

* DNA binding proteins bind to the DNA and carry out a function
* functional part which binds the DNA -- can interact with the sugar-phosphate backbone or hydrogen bond to the bases

Operons

* control transcription in bacteria

Inducible Operon

* Normally off

Repressible Operon

* normally on

Cis-operating factors

* affect activity on the same DNA molecules in which they reside
* each copy of a gene has its own promoter region as well as coding sequence
* if you have two copies → one copy promoter mutated

Trans-operating factors

* include molecule that bind to the regulatory sequence: activators proteins can increase binding polymerase to the DNA
* can be made by genes on the main chromosomes, then diffuse over to affect activity of genes that are contained in the plasmid or vie versa

Lac Operon

* negative inducible operon
* contains lacZ, lacY, and lacA
* lacI gene → encodes lac repressor → binds to operator and prevents transcription (trans- acting factor)
* if lactose present → converted to allolactose → allolactose binds to the repressor and prevents it from binding to the operator → transcription occurs and proteins are made

lacZ

* encodes beta-galactosidase → breaks lactose in glucose and galactose

lacY

* encodes permeate → transports lactose into cell

lacA

* encodes transacetylase

Mutations in LacI

LacI- → repressor not working

LacI + → functioning normal . 1 copy of normal Lacl was sufficient to restore function because trans acting element

LacIs → superrepressor. produce defective repressors that cannot be inactivated // remain bound to operator even in the presence of allolactose // dominant over normal LacI+

Mutations in LacO

LacOc→ constitutively active. operator can not bind the repressor. can’t stop transcribing B-Gal

LacO- → operator has a mutation that prevent it from binding the repressor

Mutations in LacP

LacP- → prevent RNA polymerase from binding to the promoter. Can’t produce enzymes because they can’t get RNA to function on the strand

Mutations in LacZ and LacY

LacZ- → amino acid mutations in the B-Gal gene. Prevent proper folding or function of the protein

LacY- → Amino acid mutations in the permeate gene . Prevent proper folding or function of the protein

Catabolite Repression

* when glucose present , genes that participate in metabolism of other sugars are repressed
* High glucose = little cAMP
* Low glucose = high cAMP
* cAMP binds to CAP which binds to a site near the lac operon
* lot of glucose → little cAMP → little cAMP and CAP complexes made → less CAP binding to LAC operon → less Lac operon function

Trp Operon

* functional in E. coli
* negative repressible operon
* group of genes that encode for the synthesis of amino acid tryptophan
* expressed when tryp levels are low
* repressed when tryp levels are high

Trp operon sequence regions of interactions

* Region - 1 → in the leader sequence → contains two tryptophan codons
* Region 3 → can form a stem loop structure by binding to region 2 or 4 but not both
* Binding between 2-3 → allows transcription of the structural genes → happens when low tryp
* Binding between regions 3-4 inhibits transcription → happens when high tryp

Antisense RNA

* bind to the complementary mRNA sequence and inhibit translation

Riboswitches

* regulatory sequence in the mRNA that can bind other molecules that can influence mRNA secondary structure which can impact translation
* typically located in the 3’ UTR of mRNA

DNA Packaging

Chromatin configurational Changs to make the promoter region more/less accessible to transcription factors

Transcription Initiation

Interactions between regulatory sequences and transcriptional activators/inhibitors

RNA Processing

* Alternative Cleavage and splicing of pre-RNAs

RNA Stability

Poly-A tail and interfering RNAs regulate RNA longevity

RNA Interference

RNA silencing and transcription regulation