Biochem Exam 2

Adenine

Guanine

Cytosine

Thymine

Uracil

DNA strand orientation 

antiparallel, asymmetric with a minor and major groove

Major groove

presents opportunities for sequence specific interactions to occur from the outside without unwinding 

Alternate DNA geometries

sugar pucker, syn/anti position of nucleobase

Hoogsteen base pairs

allows ¾ strands to be present in helix, found in damaged DNA and DNA bound by drugs 

RNA folding

can form base pairs with itself to form secondary structures

Melting dsDNA

seperating 2 strands

Melting temperature

temperature at which half of dsDNA is denatured and is a useful measure of dsDNA stability

Linking number

sum of writhe and twist

Changing twist →

supercoiling

Topoisomerases

change linking number of DNA by cutting, rearranging, or resealing structure

Topoisomerases effect on DNA

help DNA compaction and dealing with disruptive structures

Nucleosome

DNA wrapped around histone proteins

Inacessible DNA

packing DNA into chromatin

Histone acetylation 

DNA more acessible 

Histone methylation

DNA less acessible

Chemical mechanism of DNA synthesis

base activation of 3’ OH for nucleophilic attack on 5’ P of dNTP

Synthesis direction

5’ to 3’

Polymerases

use metals to facilitate 3’ OH attack and stabilize negatively charged phosphates 

DNA Polymerase requirement

short stretch of existing dsDNA

Primer

short piece of DNA annealed to template, facilitates addition of complimentary base pairs to growing strand

Polymerase error rate

1 in 10,000 to 1 mil base pairs

Tautomeric forms and wobble pairs →

lead to addition of non-complimentary nucleotide

Mistakes effect on DNA strand

growing strand gets passed into 5’ to 3’ exonuclease

Fixing mistakes

errogenous base gets removed and DNA polymerase gets second chance to add correct base 

DNA repilication start site

starts at origin sequence and replication forks advance in opposite directions

Helper enzymes

help unwinding and synthesis of DNA at replication fork

Leading strand

undergoes continous sequences

Lagging strand

made of serious discontinuous okazi fragments that are sealed by DNA ligase

Telomerase

prevents shortening at end of lagging strand

DNA polymerase direction

3’ to 5’

Mismatch repair pathway

corrects against misincorporation, distinguishes parental strand from daughter based on DNA methylation

Alkylation 

addition of methyl or ethyl that disrupts H bond that forms base pair 

Deamination

removal of amine, replaced by carbonyl

Depurination

removal of entire purine base

UV induced modifactions

dimerization

Direct repair

sacrifices a protien to undo a DNA alkylation event 

Base excision repair

damaged bases are removed to generate abasic sites which are then repaired 

Nucleotide excision repair

used to remove larger stretches of damaged DNA like dimers 

Homologous recombination 

repairs double strand breaks, occur between highly similar sequences

Testing homology

generating single stranded 3’ overhangs and sampling for base pairing interactions 

Polymerase function in homologus recombination 

extends 3’ overhangs using the target as a template 

Holliday junctions

4 way DNA structures that can be resolved in different ways to produce different gene products

Homologous recombination affect on diversity

increases chromosome diversity by exchanging portions of mom and dad chromosomes 

Sequence specific recombinasaes

mediate recombination at defined sites

Recombination can result in →

insertion/excision or inversion

Tyrosine recombinases

catalyze sequential single strand cleavages and proceed through a holliday junction 

Serine recombinases

catalyze simultaneous double stranded breaks, rotate DNA fragments and ligate them together 

Transposases

mobile DNA elements that copy/paste or cut/paste themselves from one location in the genome to another 

Transposase activation

lead to mosaic genotypes where different cells harbor different alleles

2 classes of transposase

retrotransposons, DNA transposons

Retrotransposons

copy/paste, RNA intermediate

DNA transposons

cut/paste, no intermediate

Transposons

can generate phenotypic variations if insertions/excisions modulate gene expression 

RNA polymerase

catalyzes RNA synthesis using DNA template, doesn’t require a primer, seperate helicase, or exonuclease  

RNA polymerase product

single strand RNA copy of coding strand, using template strand 

Promoters

position RNA polymerase to initiate at a defined location

2 Prokaryote mechanisms for termination

Rho dependent or independent

Rho-dependent

relies on helicase

Rho- independent

relies on hairpin structure

Lac operon

expresses lac genes when LacI repressor and lactose are present

Lac operon mechanism

glucose inhibits production of cAMP, cAMP increases as glucose decreases, cAMP binds to CAP, CAP binds to promoter and stimulates RNAP to activate transcription

Activator and repressor interactions

make sequence specific interactions with DNA

Sequences read through →

side chains that h-bond with DNA bases

DNA binding activity

DNA not melted, activity seperate from regulatory activity

Epigenetics

changes in gene expression not caused by changes in DNA sequence 

Chromatin

organized into accessible (euchromatin) and inacessible (heterchromatin) regions

Euchromatin

characterized by histone acetylation 

Heterochromatin

characterized by histone methylation

DNA methylation

heritable type of epigenetic regulation

5 methylcytosine

silences gene expression

Pre-mRNA

composed of introns and exons

Spliceosome

removes introns to produce mature mRNA

Alternative splicing

produces different combinations of exons and different protein isoforms

Modifications of pre-mRNA

5’ cap and 3’ poly A tail

Self splicing introns

segments of RNA that can splice themselves out of RNA polymer without assistance of proteins 

Group 2 introns

removed by mechanism similar to spliceosome

Micro RNA

small 22 nt RNA sequences that down regulate gene expression, encoded in genome

Micro RNA processing

processed in multiple steps and assemble with proteins to form RNA silencing complex

Precise pairing

cleavage of target RNA, reversible

Imprecise pairing

represses gene expression without cleavage, reversible

Making proteins

ordering of nucleotides specifies order of amino acids

Genetic code

mapping between 3 nt codons in mRNA and 20 amino acids

tRNAs

biochemcial adapters that are charged to link codons to specific amino acids

Open reading frame

continous sequence of codons flanked by start and stop codon specifying gene’s protein sequence

Ribosome

ribonucleoprotein machine that organizes polypeptide sequences using mRNA/tRNA interactions

3 interaction sites for mRNA/tRNA

A, P, E

Start codon

initiates translation that is recognized in different ways by prokaryotes and eukaryotes 

Ribosome mechanism 

N terminal amine of A site attacking ester linkage of C terminal polypeptide to P site, transferring polypeptide to tRNA in A site

Translocation of tRNA

A to P to E to gone

Termination

release factor recognizes stop codon

Translational control

provides a means of regulating gene expression

Prokaryotic translation

translation and transcription are coupled

Eukaryotic translation 

assembly of initiation complex through 5’ cap and 3’ poly A tail creates range of regulatory possibilities