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Biochemfinal cornell

TermDefinition
Central Dogma DNA->RNA->PROTEIN
DNA replication, transcription and translation all take place in the same space at the same time PROKARYOTE
Compartmentalization of transcription and translation EUKARYOTE
PURINES ADENINE + GUANINE
PYRIMIDINES CYTOSINE, THYMINE +URACIL
5'-3' ENDS PHOSPHATE- HYDROXY
DNA VS RNA 2 STRANDS VS 1 STRAND THYMINE VS URACIL N-GLYCOSIDIC BONDS OF DNA ARE SUSCEPTIBLE TO ACID HYDROLYSIS 2'OH OF RNA IS SUSCEPTIBLE TO ALKALI HYDROLYSIS
NUCLEASE CLEAVES PHOSPHODIESTER BOND (Rnase or Dnase)
RESTRICTION ENDONUCLEASE CLEAVE AT SPECIFIC INTERNAL BASE SEQUENCES
G-?-C 3 H BONDS
A-?-T(U) 2 H BONDS
A FORM OF DNA right-handed, short and broad, 2.6 A, 11 bp per turn. Formed in dehydrated DNA fibers, RNA/DNA hybrids, and RNA/RNA duplexes. 2’OH of ribose causes puckering of sugar
B FORM OF DNA right-handed, longer, thinner, 3.4 A, 10.5 bp per turn. Natural form of DNA found in prokaryotes and eukaryotes
Z FORM YOUR DRUNK DNA GO HOME LEFT HANDED, ~18A, 12 bases/turn only occurs in synthetic super high salt concentrations in runs of GC UNLESS most of the C's are methylated, then will form Z at physiological conditions
HOOGSTEEN PAIRING WEIRD FUCKING 3-STRANDED HELIX (formed by mirror repeats) rare to form (temp, ph, strand osmolarity dependent)
G4 DNA GUANOSINE TETRAPLEX DNA, SUPER STABLE, WITHSTANDS BOILING
2 FACTORS (of dna) AFFECTING MELTING TEMP G-C CONTENT, LENGTH
DNA HYBRIDIZATION measure of the relatedness between species
HUMAN GENOME SIZE 3200 MB
# OF GENES (HUMAN) 25,000
GENE DENSITY OF HUMANS 1/100 KB
# HUMAN CHROMOSOMES 23
"SHOTGUN" SEQUENCING APPROACH (VENTER) Requires >10x coverage to sequence 90 % of genome. Possible only if cost of sequencing is low and computer software is sophisticated.
"SYSTEMATIC" SEQUENCING APPROACH (COLLINS) 1 DNA IS DIGESTED, FRAGMENTS INSERTED INTO BACS 2. (LONGEST STEP) CONTIGS ARE MAPPED 3.BAC FRAGMENTS SEQUENCED 4 SEQUENCE OVERLAPS REVEAL OVERALL SEQUENCE
HUMAN GENOME SEQUENCE TYPES TRANSPOSONS=64.7%, PROTEIN CODING GENES= 1.5%, INTRONS= 25.9%
EXON PROTEIN CODING SEQUENCE OF EUKARYOTIC RNA, often encodes a single domain of a larger, multidomain protein.
INTRON eukaryotic RNA sequence removed during post transcription
HAPLOTYPE – groups of SNPs close to each other that are inherited together are compiled into haplotypes. Linkage of haplotypes with diseases can be used to map disease genes
TAG SNP a subset of SNPs that define the entire haplotype. By sequencing just these tag positions in human populations, can identify which haplotypes are present in each individual
SNP SINGLE NUCLEOTIDE POLYMORPHISM (lots of variation in humans)
CHIMP CHROMOSOME 3.29x109 bp (VS 3.2), 96% identical genome sequences, one extra pair of chromosomes (2p + 2q) corresponding to the two arms of the human chromosome 2 (BARRIER TO REPRODUCTION WITH THEM)
MESELSEN- STAHL DENSITY EXCHANGE EXPERIMENT HEAVY DNA+ NORMAL NUCLEOTIDES =SEMICONSERVATIVE DNA REPLICATION
ARTHUR AND TOM KORNBERG DISCOVERED DNA POL 1,2 +3
DNA POL 1 1. 5’-3’ DNA dependent DNA polymerase activity (CANNOT INITIATE) 2. 3’-5’ exonuclease activity or proof reading function 3. 5’-3’ exonuclease activity 4. 10-20 nucleotides polymerized/second 5. 1 subunit
IS POLYMERIZATION THERMODYNAMICALLY FAVORABLE? YES
Shape selectivity TEMPLATE STRAND BINDING TO DNA POL 1 INDUCES CONFORMATIONAL CHANGE ENSURING BASE PAIR FIDELITY
3’/5’ exonuclease activity of dna pol 1 Proof reading improves accuracy by 10^2-10^3 folds. Repair mechanisms improves accuracy by another 10^2-10^3 folds.
5’/3’ exonuclease activity of DNA POL 1 Pol&I can resynthesize a stretch of DNA by chain elongation at the nick and removing nucleosides ahead to clear the way for polymerization.
DNA POL 2 1. 7 subunits 2. 3'-5' exonuclease proofreading 3. no 5'-3' proofreading 4. 40 nucleotides/second
DNA POL 3 1. more than 10 subunits 2. 3'-5' proofreading 3. no 5'-3' proofreading 4. 250-1000 nucleotides/second (highest)
PRIMASE creates short RNA primer strands during DNA replication
REPLISOME HELICASE, PRIMASE, DNA POL1, DNA POL 3, DNA LIGASE, TOPOISOMERASE 2
OriC E. coli replication origin secquence
Dna A protein Recognizes OriC sequence, initiates replication bubble (@ AT rich region, by overwinding adjacent region)
Helicase (DnaB) unwinds DNA in replication
DnaC protein assists helicase with unwinding
SSB single stranded binding protein, necessary to keep DNA from rewinding during replication
DNA gyrase (topoisomerase 2) relieves torsional strain during DNA replication
Dam Methylase methylates GATC sequence necessary for mismatch repair in E. coli
SUPERCOILNG WHEN (in B form) Dna is wound more or less than 10.5 bp/turn
LINKING NUMBER # of times one strand of DNA winds around the other (L) L = T + W
TWISTING NUMBER (# of turns resulting from base-pairing in B form DNA or # of bp divided by 10.5 bp (Lo) T in: L=T+W
WRITHING NUMBER # of superhelical turns (W) L=T+W
TOPOISOMERS different forms of a DNA molecule that differ only in their topological property such as linking number.
TOPOISOMERASE introduce or remove supercoils by increasing or decreasing the linking number
TYPE 1 TOPOISOMERASE IN E.COLI relaxes DNA by removing negative supercoils (TOPO 1+3)
TYPE 2 TOPOISOMERASE IN E. COLI TOPO 2 (GYRASE): introduce negative supercoils (ANTIBIOTIC TARGET)( REQUIRES ATP) TOPO4: decatenates daughter DNA molecules at the completion of DNA replication
EUKARYOTIC TYPE 1 TOPOISOMERASE (1+3) relaxes negative OR positive supercoils
EUKARYOTIC TYPE 2 TOPOISOMERASE relax both positive AND negative supercoils (IIa+IIb)
POSITIVE SUPERCOILING TOPOISOMERASE STOP. THIS IS WRONG. THIS MAKES NO SENSE AND DOES NOT EXIST IN NATURE OR EVER. WE DONT LIKE POSITIVELY SUPERCOILED DNA. GO HOME.
NUCLEOSOMES unit of organization of chromatin = one bead plus adjoining DNA that leads to the next bead or 200 bp DNA plus 2
HISTONE protein core of nucleosome with DNA wrapped around found in an octomer form
amino-terminal tail NH2 terminal tails of one nucleosome extrude from the paticle and interact with adjacent nucleosomes, helping to define higher order DNA packaging. highly conserved across species
SMC structural maintenance of chromosome proteins
CONDENSIN introduces positive superhelical tension into DNA in an ATP-hydrolysis-dependent manner (SMC)
COHESIN (SMC) protein complex that regulates the separation of sister chromatids during cell division
MISMATCH REPAIR POL 3 (10^2-10^3 IMPROVEMENT) 1. MUTL-MUTS BINDS TO MISMATCH 2. MUTH BINDS TO MUTL AND NEAREST METHYLATED GATC SEQUENCE 3. MUTh CUTS UNMETHYLATED STRAND 4. DNA POL 3 TRIES AGAIN
EXCISION REPAIR damage occured throughout lifetime- POL 1
BASE EXCISION REPAIR POL 1- removal of damaged base specifically uracil or depurinated bases
NUCLEOTIDE EXCISION REPAIR POL 1- - repairs damage due to environmental mutagens such as ultraviolet rays
Direct repair DNA photolyases (not present in placental mammals) Photoreactivation of cyclobutane pyrimidine dimers induced by UV + methyltransferase repairs alkylated bases
ERROR PRONE REPAIR (BACTERIA) Pol IV and Pol V translesion DNA synthesis (reduces fidelity to one error in ~1000 nucleotides) AT REPLICATION FORK via recombination
PYRIMIDINE DIMER DNA ERROR caused by uv light, fixed by direct repair by dna photolyases
HOMOLOGOUS RECOMBINATION genetic exchanges between any two DNA molecules (or segments of the same molecule) that share an extended region of nearly identical sequence. ,
3 FUNCTIONS OF HOMOLOGOUS RECOMBINATION 1. contributes to the repair of several types of DNA damage 2. a transient link between chromatids that promotes an orderly segregation of chromosomes (EUKARYOTES) 3. enhances genetic diversity in a population
RecBCD helicase/nuclease generates a 3’ end single strand when it reaches a Chi site
BACTERIAL HOMOLOGOUS REPLICATION AS DNA REPAIR 1. 5' ND PROCESSING 2. STRAND INVASION 3. BRANCH MIGRATION 4. HOLLIDAY INTERMEDIATE RESOLUTION +LIGATION
SITE SPECIFIC RECOMBINATION RECOMBINATION LIMITED TO SPECIFIC SEQUENCE (20-200 BP) duh
HOLLIDAY INTERMEDIATE mobile junction between four strands of DNA found during homologous genetic recombination
NONHOMOLOGOUS RECOMBINATION ALLOWS THE MOVEMENT OF TRANSPOSABLE ELEMENTS
2 FACTORS AFFECTING NUMBER OF RESTRICTION ENZYME CLEAVAGE SPOTS 1. A/T:G/C RATIO 2. THE RECOGNITION SEQUENCE AND ITS LENGTH
4 RESTRICTION ENDONUCLEASE USES 1. RECOMBINANT DNA 2. CLONING LARGE DNA SEQUENCES (GENOME SEQUENCING) 3. ASSEMBLING GENOMIC SEQUENCES 4. DNA SEQUENCING
BAC bacterial artificial chromosomes- a cloning vector 100-300kb sequences, incorporated with electricity
Bacteriophage λ cloning vector high yield ~100 phage/cell
YAC yeast artificial chromosome, Used for cloning DNA segments of up to 2,000 kb. Important for the human genome sequencing project, less rearranging than BAC
STS sequence tag sites used for ordering clones in a dna library
ETS expression sequence tag used for ordering clones in a dna library
dDNA dideoxynucleotides, used in DNA sequencing, forces termination of sequence (no OH to continue)
AUTOMATED SEQUENCING REACTIoN using 5' radioactively labeled dDNA, can resolve 600-750bp
PCR polymerase chain reaction 1. heat target sequence (90C) to denature 2. add synthetic oligonucleotide primers, cool 3. add thermostable DNA pol 4. repeat- amplifies 10^6 fold
DNA GENOTYPING fingerprinting, forensic use using PCR to analyze multiple STR sequences that together are a unique profile
CODIS combined DNA index system contains >7mil STR sequences for DNA genotyping comparison
PHOTOLITHOGRAPHY This technique for preparing DNA microarray makes use of nucleotide precursors that are activated by light, joining one nucleotide to the next in a photoreaction.
tRNA transfer RNA, Between 73 and 93 bases, Contain many unusual bases, L-shaped, Half base-paired (A form)., unpaired regions that provide the structural diversity so that the tRNAs can be uniquely distinguished, 5’ end is phosphorylated, Amino acid is
AMINOACYL TRNA SYNTHETASE Read mRNA codons, translate to amino acids. each one is responsible for 1 amino acid, multiple codons
DEGENERATE CODE some amino acids are coded for by multiple codons, instead of initiating a stop sequence. minimizes deleterious effect of mutation
WOBBLE HYPOTHESIS Synonymous codons differing in the third base read for same AA, wobble has rules, hypothesis predicts the number of codons recognized by tRNA
RIBOSOME 50s+30s, 23s is not protein, catalyst property
16s rRNA used to differentiate archea/bacteria/eukaryota/ lots of differenciation
AMINO ACID ACTIVATION step 1 of protein synthesis proofread before and after by aa-tRNA sythetase very accurate
PROTEIN SYNTHESIS INITIATION STEP 2 small subunit binds mRNA and initiator aminoacyl tRNA large subunit binds requires GTP hydrolysis
SHINE-DELGARNO SEQUENCE BINDS 16s rRNA before start codon in prokaryotes
EUKARYOTIC INITIATION COMPLEX (translation) requires 5'cap and 3'polyadenylation of mRNA to initiate eLF's bind to 5' cap and 3' tail to bind to ribosome
PROTEIN SYNTHESIS ELONGATION 1. bind 2nd aminoacyl tRNA 2. form 1st peptide bond with peptidyl transferase 3. translocation (gtp hydrolysis)
PROTEIN SYNTHESIS TERMINATION 1. stop codon enters ribosome A site 2. RF1/2 bind (RF3 promotes) 3. peptidyl transferase hydrolyzes, releasing peptide
POLYSOME ribosome aggregates translating same protein concurrently
PUROMYCIN resembles aminoacyl end of tRNA, terminates peptide chains prematurely. ANTIBIOTIC
REVERSE TRANSCRIPTASE ONLY IN RNA VIRUSES. tRNA primer from previous host is required transcribes RNA to DNA to create RNA:DNA hybrid that can be transcribed by host polymerase degrades the RNA in DNA:RNA hybrid HIV therapy target high error rate (bad for curing/immunity
AZT azido di-deoxy-thymidine HIV reverse transcriptase inhibitor
DDI dideoxyinosine HIV reverse transcriptase inhibitor
TELOMERASE special eukaryotic reverse transcriptase transcribe telomeres backwards to add buffer DNA (chromosomal ends)
mRNA 2-5% of all RNA short lived, heterogeneous
tRNA stable, ~15% of all RNA
rRNA ~80% of all RNA, stable
SPIEGELMAN EXPERIMENT determined makeup/proportions of RNA
SECONDARY RNA STRUCTURES BULGE, INTERNAL LOOP, SINGLE STRAND, HAIRPIN
4 STAGES OF TRANSCRIPTION 1.RNA pol recognizes promoter region 2. moves along DNA synthesizing RNA 3. stops at termination site 4. releases RNA
SIGMA 70 binds to DNA promoter region in order fo RNA POL to bind, released during transcription TATA BOX/ 10/30 sequence
RHO helicase separates RNA from RNA pol in P-dependent termination
INTRON NONCODING MRNA SEQUENCE IN EUKARYOTES REMOVED DURING PROCESSING
5' MRNA CAP 7-methylguanosine cap, added to 5'end of mRNA while still being synthesized
SPLICEOSOME attached to RNA pol, removes introns, splices exons
POLY (A) TAIL added to transcribed mRNA sequences when polyadenylate polymerase recognizes bound polyadenylation factors
lac OPERON REGULATION 1. presence of lactose removes the repressor, allowing lactase transcription 2. lack of glucose activates cAMP, activating promoter which promotes high lactase transcriptase
DNA FOOTPRINTING IDENTIFY PROMOTER BINDING SITE - THE DNA SEQUENCE WHERE A PROTEIN BINDS.
# OF PROTEINS IN HUMANS 60,000-100,000
Created by: zideutsch on 2013-05-14



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