biochem kms

it is 2:15 am

intercalating agents structure

hydrophobic, aromatic, heterocyclic rings

intercalating agents examples

ethidium bromide, acridine orange, actinomycin D, sybr green

DNA poly III functions

5—>3 = DNA polymerase, 3—>5 = exonuclease and proofreading

DNA poly I functions

5—>3 = DNA polymerase and exonuclease to remove primer

Ciproflaxin and Quinolones

antimicrobial agents that act on topoisomerase II

difference between type I DNA topoisomerase and Type II DNA topoisomerase

type I is for more linear DNA, type II is for supercoils

Rnase H funciton in euks

removes rna primers

Anticancer drugs that work on topoisomerase I

camptotehcin, indenoisoquioline

anticancer drugs that work on human topoisomerase II

etoposide

components of chromatin

DNA, histone

histone octamer parts

(H3)2(H4)2 tetramer, (H2A-H2B) dimers x2

Reverse transriptase 3 activities

RNA-dependent DNA poly, ribonuclease H, DNA-dependent DNA poly

nucleoside analogs put a modification on the () group at ()’c end of deoxyribose

hydroxyl, 3’

Cisplatin

links 2 purines, leads to cell death by replacing chlorides of adjacent purines (guanine) with nitrogens

mismatch results from

lack of proofreading

how many damaging events does each cel undergo per day

>20,000

how many replication errors per cell per day

>10,000

Most common base alteration in cancer cells + mechanism

C to T via methylation then deamination with cytosine deaminase

Function of ROS

induces DNA damage

ROS example

hydroxyl radical

8-oxoguanine story

guanine oxidized to become 8-oxoguanine, which can bp to adenosine not cytidine

most common type of alkylation base alteration

methylation

depurination mechanism + result

glycosidic bond broken, makes AP site which gets misread

what causes more dna damage?

deamination, oxidation, methylation depurination » mutagenic agents

Thymine dimer comes from

UV radiation, leads to crosslinking of adjacent thymines

double strand break comes from

ionizing radiation (xrays, radioactive decays), free radical products of oxidative metabolism

Rb E2F story

Rb is bind to E2F for G0, beginning of G1, Rb will get phosphorylated as a result of Cyclin-cdk and ATP and leave at late G1, letting transcription happen (goes to S phase)

What does Rb E2f block?

transactivation domain

p53 —> P21 story

DNA damage causes activation of ATM/ATR, activates p53, activates p21, inhibits cdk2/cyclin E —> G1 arrrest

DNA reversal repair

does not need a template, no breaking phosphodiester bond

DNA reversal repair example

methylation of guanine reversed by methyl guanine methyl transferase

general DNA repair steps

1) recognize bad bases 2) remove bad bases 3) repair gap with dna poly, ligase

Base excision repair enzymes used (in order)

glycosylase, AP endonuclease, excision endonuclase, polymerase, DNA Ligase

Nucleotide excision repair enzymes used (in order)

excision endonuclease, polymerase, ligase

What is HNPCC associated with?

mutations in DNA mismatch repair pathway

Xeroderma pigmentosum associated with

Nucleotide excision repair (7+ genes)

Ataxia telangiectasia associated with

response to dsDNA breaks

Breast cancer + ovarian cancer associated with

germline transmission of mutant BRCA1/BRCA2 allele, ovarian > breast for incidence within families

Cytochrome p450 does…

conversion of procarcinogens to carcinogens

how does cigarette smoke become carcinogen?

compound benzo[a]pyrine gets oxidized, epoxide added, covalently binds DNA

how does aflatoxin b1 become carcinogen?

adds expoxide by cytochrome p450, covalently binds DNA to make DNA adduct

examples of procarcinogens

cigarette smoke, aflatoxin B, vinyl chloride, styrene

Ames test procedure

mix compound with rat liver enzyme, add salmonella that can’t grow on histidine, count bacterial colonies

why is rat liver added to ames test?

mimic mammalian metabolism

gain-of-functions

conversion of proto-oncogene to oncogene gives abnormal stimulation of cell cycle

oncogene results

fucked proteins

Tumor suppressor genes

prevent uncontrolled cell growth by repairing damaged DNA, control cell adhesion, stop cell cycle in cell signaling pthwy

RNA chains can be initiated de

novo

Sense (coding) vs Antisense (template) strand

RNA builds 5→3, goes on antisense strand but sense is on the other side

RNA polymerase requirements

templsate, activated precurors, divalent metal ions

parts of holoenzyme with function

sigma - initiation, core - elongation, beta - termination

sigma subunit use 

helps locate promotor sites, decreases affinity of polymerase for DNA to help find fast, help identify, dissociates from enzyme when done

Promoter sequences in E coli

-10 pribnow box (TATAAT), -35 sequence

initiation of prokaryotic transcription

binding RNA polymerase holoenzyme to promoter region

Prokaryote elongation

use ribonucleoside triphosphate, release pyrophosphate

Prokaryotic termination

RNA poly stops moving, transcript falls off from transcription complex

rho-independent (intrinsic) termination

rna transcript makes stable hairpin turn w string of Us

rho-dependent termination

rho factor bind a C-rich reagion, contains helicase activity to displace DNA template strand

Eukaryotic gene promoter

RNA poly 2

TATA, CAAT, GC boxes recognized by

not RNA polymerase

where is TATA box

25 nucleotides up

CAAT box located

40-150 nucleotides up

typical protein endocing gene elements

enhancers, promoter, tss, exons, introns, polya signal seq, transctiption termination region

posttranscriptional procesing of rrNa include

removal of external transcribed spacers, internal transcribed spacers

what acts for precursor of 3 types of rRnas

45s —> 28s, 5.8s, 18s

Cleavage posttranscriptional mod

sequence at 5’ end cleaved by RNAse P

Addition posttranscriptional mod

tRNA nucleotidyl transferase adds to 3’ end

modified nucleosides posttranscriptional mod

methylation, reduction, transversion deamination

methylation of A, G

mA, mG

reduction of U

DHU

transverson of U

psi

deaminaiton of A

I

Ribozymes features

in nature happen in sel-splicing intron + RNA encoded parasies, ribosomes limited to cleavage and ligation, catalytic efficiency usually much lower

posttranslational modification types

5’ cap, 3’ polyadenylation, intron removal

5’ capping how to

7-methyl guanosine cap added to 5’ end via 5’-5’ phosphate linkage

5’ capping purpose

permit initiation of translation, stabilize mRNA

3’ polyadenylation how to

added to 3’ end of transcript

3’ adenylation purpose

stabilize mRNA, exit of it from nucleus

2 ways to kill introns

cleavage via special splicing endonuclease and ligation with ligase activities (ribozyme), 2-step rxns with spliceosomes

Splicing enzyme are…

snRNPs

snRNPs recognize…

splice sequence, where intron starts with GU and ends with AG

siRNA vs miRNA

siRNA does viral replication +uses dicer (endoribonuclease that cuts up RNA), miRNA usses RNA poly II

siRNA base pairing

double strand RNA perfectly matched, only targets at mRNA, natural antiviral defense in plants fungi invertebrates

miRNA base pairing

single strand DNA with some matched but >1target coding region, mechanism of regulation of gene expression

Rifampin how to

blocks transcription initiation by binding to free poly, inhibits elongation by binding to DNA bound polymerase

Actinomycin D how to

intercalats btwn bases, prevents DNA from being used as template

Alpha Amantin how to

high specificity for euk RNA poly II, synthesized by a mshroom

hemoglobin mckees rocks

nonsense mutation, tyrosine made to a stop codon

wobble hypothesis

i can bind to u c a, g can bind u or c, u can bind a g

which base determines degree of wobble?

first

aminoacyl-tRNA synthetase

attach AA to tRNA, activated by reacting with ATP to make aminoacyl-amp, activated amino acid transferred from aminoacyl-amP to tRNA, makes aminoacy-tRNA, uses 2 high energy phosphates

fidelity of translation maintained by

synthetase (editing activity)

Initiation of translation in proks

binding of mRNA to 3’ end of 16srRNA in 30s subunit

where does initiation of translation begin in bacteria?

shine dalgarno sequence - 25 nucleotides from 5’ end, directs protein synthesis machinery to start site and positions AUG in p site

termination of translation release factors

RF1 (UAA), RF2 (UGA and UAA), RF3 bind gtp and erf

numbers of translation termination

cleavage of 4 high energy bonds req per amino acid addition to polypeptide

preinitiation complex of translation in euks

40s ribosome, met-tRNA in association with eIF-2

euk translation initiation 2 3 4

2) elF4E binds to 5’ cap, facilitates binding of PIC to mRNA 3) PIC binds to 5’ end of mRNA, moves 3’ searching for AUG 4) adding 60s subunit to make 80s initiation complex

SRP functions

inhibit translation, docks ribosome on ERE

what affects initiation and how?

streptomycin interferes with binding of fMet-tRNAf (N-formyl-met in bacteria), inhibits protein synthesis initiation in bacteria