Biochemfinal cornell
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| Central Dogma | DNA->RNA->PROTEIN
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| DNA replication, transcription and translation all take place in the same space at the same time | PROKARYOTE
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| Compartmentalization of transcription and translation | EUKARYOTE
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| PURINES | ADENINE + GUANINE
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| PYRIMIDINES | CYTOSINE, THYMINE +URACIL
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| 5'-3' ENDS | PHOSPHATE- HYDROXY
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| 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
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| NUCLEASE | CLEAVES PHOSPHODIESTER BOND (Rnase or Dnase)
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| RESTRICTION ENDONUCLEASE | CLEAVE AT SPECIFIC INTERNAL BASE SEQUENCES
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| G-?-C | 3 H BONDS
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| A-?-T(U) | 2 H BONDS
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| 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
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| 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
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| 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
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| HOOGSTEEN PAIRING | WEIRD FUCKING 3-STRANDED HELIX (formed by mirror repeats)
rare to form (temp, ph, strand osmolarity dependent)
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| G4 DNA | GUANOSINE TETRAPLEX DNA, SUPER STABLE, WITHSTANDS BOILING
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| 2 FACTORS (of dna) AFFECTING MELTING TEMP | G-C CONTENT, LENGTH
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| DNA HYBRIDIZATION | measure of the relatedness between species
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| HUMAN GENOME SIZE | 3200 MB
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| # OF GENES (HUMAN) | 25,000
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| GENE DENSITY OF HUMANS | 1/100 KB
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| # HUMAN CHROMOSOMES | 23
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| "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.
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| "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
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| HUMAN GENOME SEQUENCE TYPES | TRANSPOSONS=64.7%, PROTEIN CODING GENES= 1.5%, INTRONS= 25.9%
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| EXON | PROTEIN CODING SEQUENCE OF EUKARYOTIC RNA,
often encodes a single domain of a larger, multidomain protein.
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| INTRON | eukaryotic RNA sequence removed during post transcription
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| 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
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| 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
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| SNP | SINGLE NUCLEOTIDE POLYMORPHISM (lots of variation in humans)
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| 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)
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| MESELSEN- STAHL DENSITY EXCHANGE EXPERIMENT | HEAVY DNA+ NORMAL NUCLEOTIDES =SEMICONSERVATIVE DNA REPLICATION
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| ARTHUR AND TOM KORNBERG | DISCOVERED DNA POL 1,2 +3
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| 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
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| IS POLYMERIZATION THERMODYNAMICALLY FAVORABLE? | YES
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| Shape selectivity | TEMPLATE STRAND BINDING TO DNA POL 1 INDUCES CONFORMATIONAL CHANGE ENSURING BASE PAIR FIDELITY
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| 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.
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| 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.
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| DNA POL 2 | 1. 7 subunits
2. 3'-5' exonuclease proofreading
3. no 5'-3' proofreading
4. 40 nucleotides/second
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| DNA POL 3 | 1. more than 10 subunits
2. 3'-5' proofreading
3. no 5'-3' proofreading
4. 250-1000 nucleotides/second (highest)
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| PRIMASE | creates short RNA primer strands during DNA replication
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| REPLISOME | HELICASE, PRIMASE, DNA POL1, DNA POL 3, DNA LIGASE, TOPOISOMERASE 2
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| OriC | E. coli replication origin secquence
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| Dna A protein | Recognizes OriC sequence, initiates replication bubble (@ AT rich region, by overwinding adjacent region)
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| Helicase (DnaB) | unwinds DNA in replication
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| DnaC protein | assists helicase with unwinding
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| SSB | single stranded binding protein, necessary to keep DNA from rewinding during replication
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| DNA gyrase (topoisomerase 2) | relieves torsional strain during DNA replication
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| Dam Methylase | methylates GATC sequence
necessary for mismatch repair in E. coli
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| SUPERCOILNG | WHEN (in B form) Dna is wound more or less than 10.5 bp/turn
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| LINKING NUMBER | # of times one strand of DNA winds around the
other (L)
L = T + W
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| 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
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| WRITHING NUMBER | # of superhelical turns (W)
L=T+W
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| TOPOISOMERS | different forms of a DNA molecule that differ only in their topological property such as linking number.
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| TOPOISOMERASE | introduce or remove supercoils by increasing or decreasing the linking number
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| TYPE 1 TOPOISOMERASE IN E.COLI | relaxes DNA by removing negative supercoils (TOPO 1+3)
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| 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
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| EUKARYOTIC TYPE 1 TOPOISOMERASE | (1+3) relaxes negative OR positive supercoils
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| EUKARYOTIC TYPE 2 TOPOISOMERASE | relax both positive AND negative supercoils (IIa+IIb)
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| 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.
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| NUCLEOSOMES | unit of organization of chromatin = one bead plus adjoining
DNA that leads to the next bead or 200 bp DNA plus 2
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| HISTONE | protein core of nucleosome with DNA wrapped around
found in an octomer form
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| 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
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| SMC | structural maintenance of chromosome proteins
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| CONDENSIN | introduces positive superhelical tension into DNA in an ATP-hydrolysis-dependent manner (SMC)
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| COHESIN | (SMC) protein complex that regulates the separation of sister chromatids during cell division
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| 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
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| EXCISION REPAIR | damage occured throughout lifetime- POL 1
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| BASE EXCISION REPAIR | POL 1- removal of damaged base
specifically uracil or depurinated bases
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| NUCLEOTIDE EXCISION REPAIR | POL 1- - repairs damage due to environmental
mutagens such as ultraviolet rays
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| Direct repair | DNA photolyases (not present in placental mammals)
Photoreactivation of cyclobutane pyrimidine dimers induced by UV
+ methyltransferase repairs alkylated bases
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| 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
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| PYRIMIDINE DIMER DNA ERROR | caused by uv light, fixed by direct repair by dna photolyases
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| HOMOLOGOUS RECOMBINATION | genetic
exchanges between any two DNA molecules (or segments of
the same molecule) that share an extended region of nearly
identical sequence. ,
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| 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
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| RecBCD | helicase/nuclease
generates a 3’ end single strand
when it reaches a Chi site
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| BACTERIAL HOMOLOGOUS REPLICATION AS DNA REPAIR | 1. 5' ND PROCESSING
2. STRAND INVASION
3. BRANCH MIGRATION
4. HOLLIDAY INTERMEDIATE RESOLUTION +LIGATION
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| SITE SPECIFIC RECOMBINATION | RECOMBINATION LIMITED TO SPECIFIC SEQUENCE (20-200 BP) duh
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| HOLLIDAY INTERMEDIATE | mobile junction between four strands of DNA found during homologous genetic recombination
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| NONHOMOLOGOUS RECOMBINATION | ALLOWS THE MOVEMENT OF TRANSPOSABLE ELEMENTS
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| 2 FACTORS AFFECTING NUMBER OF RESTRICTION ENZYME CLEAVAGE SPOTS | 1. A/T:G/C RATIO
2. THE RECOGNITION SEQUENCE AND ITS LENGTH
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| 4 RESTRICTION ENDONUCLEASE USES | 1. RECOMBINANT DNA
2. CLONING LARGE DNA SEQUENCES (GENOME SEQUENCING)
3. ASSEMBLING GENOMIC SEQUENCES
4. DNA SEQUENCING
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| BAC | bacterial artificial chromosomes- a cloning vector
100-300kb sequences, incorporated with electricity
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| Bacteriophage λ cloning vector | high yield ~100 phage/cell
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| 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
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| STS | sequence tag sites
used for ordering clones in a dna library
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| ETS | expression sequence tag
used for ordering clones in a dna library
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| dDNA | dideoxynucleotides, used in DNA sequencing, forces termination of sequence (no OH to continue)
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| AUTOMATED SEQUENCING REACTIoN | using 5' radioactively labeled dDNA, can resolve 600-750bp
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| 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
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| DNA GENOTYPING | fingerprinting, forensic use
using PCR to analyze multiple STR sequences that together are a unique profile
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| CODIS | combined DNA index system
contains >7mil STR sequences for DNA genotyping comparison
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| 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.
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| 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
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| AMINOACYL TRNA SYNTHETASE | Read mRNA codons, translate to amino acids. each one is responsible for 1 amino acid, multiple codons
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| DEGENERATE CODE | some amino acids are coded for by multiple codons, instead of initiating a stop sequence. minimizes deleterious effect of mutation
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| 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
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| RIBOSOME | 50s+30s, 23s is not protein, catalyst property
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| 16s rRNA | used to differentiate archea/bacteria/eukaryota/ lots of differenciation
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| AMINO ACID ACTIVATION | step 1 of protein synthesis
proofread before and after by aa-tRNA sythetase
very accurate
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| PROTEIN SYNTHESIS INITIATION | STEP 2
small subunit binds mRNA and initiator aminoacyl tRNA
large subunit binds
requires GTP hydrolysis
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| SHINE-DELGARNO SEQUENCE | BINDS 16s rRNA before start codon in prokaryotes
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| 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
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| PROTEIN SYNTHESIS ELONGATION | 1. bind 2nd aminoacyl tRNA
2. form 1st peptide bond with peptidyl transferase
3. translocation (gtp hydrolysis)
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| PROTEIN SYNTHESIS TERMINATION | 1. stop codon enters ribosome A site
2. RF1/2 bind (RF3 promotes)
3. peptidyl transferase hydrolyzes, releasing peptide
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| POLYSOME | ribosome aggregates translating same protein concurrently
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| PUROMYCIN | resembles aminoacyl end of tRNA, terminates peptide chains prematurely. ANTIBIOTIC
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| 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
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| AZT | azido di-deoxy-thymidine
HIV reverse transcriptase inhibitor
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| DDI | dideoxyinosine
HIV reverse transcriptase inhibitor
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| TELOMERASE | special eukaryotic reverse transcriptase
transcribe telomeres backwards to add buffer DNA (chromosomal ends)
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| mRNA | 2-5% of all RNA short lived, heterogeneous
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| tRNA | stable, ~15% of all RNA
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| rRNA | ~80% of all RNA, stable
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| SPIEGELMAN EXPERIMENT | determined makeup/proportions of RNA
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| SECONDARY RNA STRUCTURES | BULGE, INTERNAL LOOP, SINGLE STRAND, HAIRPIN
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| 4 STAGES OF TRANSCRIPTION | 1.RNA pol recognizes promoter region
2. moves along DNA synthesizing RNA
3. stops at termination site
4. releases RNA
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| SIGMA 70 | binds to DNA promoter region in order fo RNA POL to bind, released during transcription
TATA BOX/ 10/30 sequence
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| RHO helicase | separates RNA from RNA pol in P-dependent termination
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| INTRON | NONCODING MRNA SEQUENCE IN EUKARYOTES REMOVED DURING PROCESSING
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| 5' MRNA CAP | 7-methylguanosine cap,
added to 5'end of mRNA while still being synthesized
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| SPLICEOSOME | attached to RNA pol, removes introns, splices exons
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| POLY (A) TAIL | added to transcribed mRNA sequences when polyadenylate polymerase recognizes bound polyadenylation factors
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| 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
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| DNA FOOTPRINTING | IDENTIFY PROMOTER BINDING SITE - THE DNA SEQUENCE WHERE A PROTEIN BINDS.
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| # OF PROTEINS IN HUMANS | 60,000-100,000
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