Unit 2 Flashcards
Nucleoside
Base connected to sugar
Nucleotide
Base + Sugar + Phosphate
Phosphodiester Bond
Btwn nucleotides
Form backbone of nucleic acids
Covalent
Glycosidic Bond
Connection btwn base and sugar
Covalent
Hydrogen Bond
Form base pairs
Non covalent
Watson Crick Franklin Pairing
Anti/Anti
C+G
A+T
Hoogsteen Pairing
Syn/Anti
Exotic RNA structures
Purines
A and G
2 rings
Pyrimidines
C, T, and U
1 ring
DNA vs RNA
DNA lacks 2’ OH group
Ester Linkage
Attach phosphate to nucleoside
A + T
2 x H-bonds
G + C
3 x H-bonds
Base stacking interactions
van der Waals (btwn base faces)
pi stacking
B form structure
Anti parallel
Right handed
Backbone on outside
Bases on inside
Major groove
Wide and deep
Minor groove
Shallow and narrow
RNA preferred conformer
C3’ endo
C2’ endo causes sterics
DNA preferred conformer
C2’ endo
T melting
Temp at which the helix is 1/2 double stranded and 1/2 single stranded
Negative supercoiling
Lk < 0
DNA = underwound
Positive supercoiling
Lk > 0
DNA = overwound
Topoisomerase
Change DNA topology
Resolve supercoiling that develops when genome is locally unwound
Nucleosome
DNA wrapped around a histone
Chromatin
Histones packaged into 30 nm fiber
Involves supercoiling
Heterochromatin proteins
Bind across methylated histones
Inaccessible
Transcription can’t occur
Marked by methylation
Euchromatin
Accessible
Transcription can occur
Marked by acetylation
Chem mechanism of nucleotide addition
Base activates 3’ OH
3’ OH does nucleophilic attach on 5’ phosphate
Synthesis direction
5’ to 3’
DNA Polymerase
Catalyze nucleotide addition
Requires stretch of primer
Polymerase and Mg2+
Mg2+ facilitates chemistry
- Activates 3’OH
- Stabilize - charge of phosphate
- Mg2+ binds to Asp
Primer
Short stretch of RNA annealed to template
Facilitates addition of complementary base pairs
Takes advantage of energy of base stacking
DNA Polymerase Mistakes (2)
Tautomerization
Wobbles
Wobbles
Normal bases that bind inappropriately
due to shift in position of nucleotides
Tautomerization
Change into form where positions of H-bond donor/acceptor changes
DNA Polymerase Activities
Add nucleotides via polymerase domain
Remove mismatches via exonuclease domain
Incorrect base detection
Destabilization of DNA structure
Adopt conformation that moves 3’ end into exonuclease site
Incorrect base correction
3’ end enters exonuclease site
Terminal nucleotide removed
Polymerase gets 2nd chance
DNA Pol III
Main replicative polymerase
Built for speed
Copies entire genome
Built for processivity
DNA Pol I
Odd jobs
Polymerizes small stretches as part of cleanup/repair
Built for accuracy
Start of replication
Origin of replication
Helicase
Melt and unwind DNA
Primase
Synthesize small RNA sequence as a primer for DNA Pol III
Leading strand
Continuous synthesis
Parent 3’ end on the bottom left
Lagging strand
Discontinuous synthesis
Parent 3’ end on the top right
Okazaki fragments
Fragments that make up discontinuous strand
Lagging strand activities
Primase adds primer
Pol III extends DNA until encounter of next fragment
Primase + Pol III make fragments
Pol I removes primer + replaces with DNA
Ligase seals fragments
Ligase
Seals nicked DNA fragments
How does Ligase work
- Ligase uses ATP to adenylate itself
- AMP transferred to 5’ phosphate of nicked strand
- Phosphate has good LG
- Base catalyzed 3’ OH attack seals strand
Single stranded binding proteins
Keep ssDNA from reannealing
Telomere
Sequence of repetitive sequences at the ends of chromosomes
Telomerase
Carries RNA fragment
Template DNA synthesis at end of telomere on parental strand
Prevent shortening of strands
Main pathways to mutation
Proofreading missed a mistake
Modified base leads to incorrect pairing
Distinguish parent from daughter strand for repair
Mark parental strand with methylation marks
Mismatch Repair Pathway
Mismatch detected b MutL-MutS
DNA threaded through complex until encounter of MutH
Complex cleaves unmodified daughter strand
Nuclease and helicases degrade unmethylated strand
Gap filled in by DNA Pol III
Sealed by ligase
Alkylation
Add methyl or ethyl groups to nucleobase
Deamination
Replace amine with carbonyl group on nucleobase
Thymine is immune
Depurination
Loss of entire purine base from nucleotide
UV light induced dimerization
Adjacent Thymines and Cytosines become cross linked into dimers
Distorted structure
Misread by Polymerase
Repair of Alkylation
Direct repair
Repair of Deamination
Base excision repair
Repair of Depurination
Base excision repair
Repair of dimerization
Nucleotide excision repair
Direct repair pathway
Alkyl groups transferred to alkyltransferase
Group is permanently attached to protein
1 Protein sacrificed per repair
Base excision repair (Depurination)
AP nuclease recognizes abasic site
Nicks at site of damage
Free 3’ OH provides template for DNA polymerase to extend
Long or short patch pathway
Ligase seals strands
Base Excision repair (Deamination)
Convert base into abasic site with glycosylase enzyme
Repair site with base excision repair mechanism
Long patch
Polymerase extend past abasic site
Old part is displaced and cleaved
Ligase seals the new strand
Short patch
Old part removed by lyase
Abasic site filled in by polymerase
Ligase seals the strand
Why is the genome DNA
RNA is more susceptible to degradation
Exonuclease
Enzyme that cleaves nucleotides one at a time from the end of a nucleic acid
Endonucelase
Enzyme that cleaves the phosphodiester backbone within a nucleotide chain
DNA Recombination
Process in which pieces of DNA are broken and reassembled to create new fragments
Homologous Recombination
Occurs between any 2 homologous sequences
Leads to exchange of DNA material
Material in between the homologous sequences doesn’t need to be the same
Sequence Specific Recombination
Very specific sequences used
Highly controlled w/ predictable outcomes
Tyrosine Recombinase
2 sequential single strand breaks
Holliday junction intermediate
Serine Recombinase
Two double strand breaks introduced simultaneously
No holliday junction
Transponsons
Mobile DNA elements that move around the genome
Insertions may or may not occur
Cause changes in gene expression of inserted into coding region
Class 1 Transponsons
Copy/paste
Sequence copied to an RNA intermediate that is used to make a second DNA copy for insertion
RNA intermediate
RNA transponson
Class 2 Transponsons
Cut/paste
DNA transposon
DNA sequence is excised and pasted elsewhere in the genome
Template Strand
Used to make RNA copy of coding strand
Coding Strand
Strand of interest that you want to copy into RNA
RNA Polymerase
Catalyzes RNA synthesis from DNA templates
Doesn’t need a primer
Catalyzes own helicase activity
RNA Pol Proofreading
No exonuclase site
Can backtrack to proofread
Active site for polymerization acts as nuclease and cleave strands
Promoter
Specifies where RNA polymerase will start transcription
Specific sequences at defined distances from intended start site
Sigma factor
In bacteria
Recognizes specific sequence of DNA that defines a promoter
Changing the sigma factor changes which sequences are transcribed
RNA pol in eukaryotes
3 forms, each with own initiation specialty
Regulated by other elements and sequences
Rho dependent termination
RNA pol transcribes through a stretch of DNA that produces a binding site for Rho factor
Rho factor = helicase
Moves towards 3’ end
Climbs RNA until it reaches RNA/DNA anneal point
Rho unwinds RNA/DNA hybrid → terminates transcription
Helicases unwind DNA hybrids (peel off RNA from DNA)
Rho independent termination
RNA polymerase transcribes through a stretch of DNA that makes
- A G/C rich region that adopts a hairpin structure (form base pairs with itself)
- A U rich segment downstream of hairpin
- Hairpin causes RNA pol to stall
U rich region has weak base pairing interaction with template strand
Stall + weak interaction cause RNA pol to fall off
Lac Operon
If lactose is absent
Repressor binds to operator;specific sequence that overlaps with promoter
RNA pol is prevented from transcribing the lactose gene
Lac Operon
If lactose is present
Lactose binds repressor and blocks DNA binding
Repressor converted to inactive form → does not bind
RNA pol can bind and initiate transcription
cAMP
Binds to CAP
Increases when glucose decreases
CAP
Binds to promoter and stimulates RNA pol recruitment (activator)
Basal machinery
Is needed to assemble RNA pol at promoter
Eukaryotic
Activators
Recruit coactivators
Bind to enhancer elements
Eukaryotic
Repressors
Block coactivator recruitment
Block activator binding to DNA
Recruit corepressors
How side chains read DNA
Hydrogen bonds form between specific amino acids and nucleotide bases
Based on patterns of donors and acceptors
Proteins can read bases without having to melt DNA
Use the major groove to read DNA
Epigenetics
Changes to gene expression not caused by change in DNA sequence itself
Chromatin Modification
Histone methylation compacts the genome
Histone acetylation opens up the genome
Histone acetylation
Acetylated on lysine residues by HATs
Removed by HDACs
Associated with active gene expression
Reduces affinity of histones for DNA
Remove positive charge on lysine
Directly recruit activators
Histone methylation
Methylated on lysines by histone methyltransferases
Removed by histone demethylases
Associated with inactive gene expression
5 methyl cysteine
Methyl mark does not interfere with base pairing
Inhibits transcription
New methylation marks added by de novo methyltransferases
Permanently silence areas of genome
mRNA
Contains info to make a specific protein sequence via translation
Intron
sequences removed from RNA transcript
Exon
sequences that remain in RNA transcript, are expressed
Splicing is performed
In the nucleus
By the spliceosome
5’ cap
7 methyl guanosine cap
Aids in formation of translation initiation complex
Protect 5’ end from degradation
Signals for export out of nucleus
3’ Poly A tail
Poly tail added by PAP
Signal sequence recruits enzyme to add tail
Stabilize mRNA
Promote translation
Signal to export out of nucleus
Reason for RNA modification
Many ways to splice mRNA
Can get different flavors or proteins from 1 mRNA strand
Processing is proof that your cells produced the mRNA
Spliceosome
Complex machine of proteins and snRNAS (non coding)
Ribonucleoprotein (RNP)
Splicing steps
2’ OH from ribose within intron cleaves backbone at 5’ splice site
Free 3’ OH of the 5’ first exon attacks phosphate connecting intron to second exon
Release lariat
Group 1 introns
Use free guanosine nucleotide to initiate cleavage
3’ OH on guanosine attack phosphate at 5’ splice site
3’ OH of exon attacks phosphodiester attack at 3’ exon
Release linear fragment as the intron
Group 2 Intron
Use adenosine to initiate cleavage
2’ OH of adenosine attacks 5’ splice site
3’ OH of 5’ exon does attack of backbone at 3’ exon
Release lariat as the intron
MicroRNA
22 nucleotide genome encoded RNAs
Regulate gene expression
Base pairing used to locate miRNA targets
miRNA perfect match
Destruction and degradation of target mRNA
Irreversible
miRNA imperfect match
Repression but not destruction
RNA stored in P-body structures
Reversible
Techniques for making synthetic DNA
Chemical synthesis of short oligonucleotides
PCR to amplify DNA fragments
Assembly of DNA fragments and cloning of synthetic genes
Techniques for reading out sequence of DNAs
Sanger sequencing
Next generation/high throughput sequencing techniques
Modifying the biology of living systems using synthetic DNA
Plasmid based gene expression systems
CRISPR/Cas9 genome modification
Translation
Going from mRNA to protein
Genetic code
Redundant
Universal
Triplet codons
tRNA
Adapter molecule that links triple codon to its associated amino acid
Amino acid held by 3’ end
Use WCF base pairing to read codons in mRNA
Addition of amino acid to tRNA
Amino acid activated by adenylation of carboxyl group
tRNA synthetase selects matching tRNA and transfers amino acid to tRNA 3’ OH
Wobble base pairs
First 2 bases create coding specificity
Third base does not make strong interaction
Does not need to be a perfect match
Reading frame
Consecutive, non overlapping codon sequences translated into a polypeptide
Missense mutation
Change mRNA sequence from a codon for 1 amino acid to a codon for a different amino acid
Silent mutation
Change mRNA sequence to a synonymous codon
Nonsense mutation
Change mRNA sequence form a codon for an amino acid to a stop codon
Terminate translation
Frameshift mutation
Disrupt normal reading frame by inserting or deleting nucleotides that are not in multiples of 3
Give entirely different sequence
Ribosome
Initiates synthesis at start codon
Forms peptide bonds between amino acids to make a polypeptide
Terminates synthesis at the stop codon
Prokaryote translation iniation
Base pairing between ribosome RNA and mRNA at shine dalgarno positions ribosome at intended start codon
Eukaryote translation initation
Scanning mechanism
Ribosome assembles initiation complex
A site
Holds tRNA with next amino acid
P site
Holds tRNA with growing peptide
E site
Holds tRNA that will exit
Translation condensation rxn
A site N amine attacks P site ester linkage to the C end
Translation direction
Occurs in N to C direction
Translation termination
Release factor (protein) recognizes stop codon and promotes peptide release + translation termination
Prokaryote translation regulation
Translation and transcription coupled
Physical connection between transcription and translation allows for clever regulatory strategies
Eukaryotes translation regulation
Block interaction between 5’ cap and capping protein
Decapp enzymes
Binding proteins can prevent formation of initiation complex
Enzymes trim or remove poly A tail
Oligonucleotides
Chemically synthesized
10-80 nucleotides
Chemical synthesis is fast, easy, cheap
Solid state
PCR
Amplify sequence of interest
Use pair of primers
Copied DNA spans btwn priming sites
Gibson assembly
Makes uses of cocktail of enzymes
Mimic recombination reaction
Produce free overhangs
Sanger method
Exploit nucleotide structure to read DNA sequence
Incorporate di-deoxynucleotide into DNA
No 2’ OH or 3’ OH
CRISPR/Cas9
Endonuclease that is programmed by RNA sequence that tells it what to cut
Use WCF base pairing between guide RNA and dsDNA to locate target
Test dsDNA makes protein/DNA interaction with a short sequence (PAM)
Extensive base pairing leads to cleavage
