M2M wk 1 Flashcards
High energy compounds
ATP, NADH, NADPH, FADH2
first law of thermo
energy is always conserved
second law of thermo
delta S(universe) always increasing
Redox rxn related to gibbs free energy
G=nFE
gibbs free energy eqn
G=RTlnKeq
high energy bonds
Thioeseter bonds (acetyl CoA; P-O-P (ATP); P-N (phosphocreatine); C-O-P (phosphoenolpyruvate)
electron flow
glucose= major source of e-, O2 is the final acceptor; the circuit is a series of proteins including cytochromes with FeII/FeIII
biological information transfer:
DNA transcription to RNA translation to Proteins
Purine bases
adenine and guanine
pyrimdine bases
Thymine (urain in RNA) and cystine
nucleotide solubility
purines < pyrimidine; bases < nucleoside < nucleotide
gout from Lesch-nyhan disease
accumulation of uric acid in joints due to a deficiency in phosphoribosyl transeferase which converts guanine to GMP (purine salvage pathway)
DNA convention
5’ to 3’ (phosphodiester bonds)
AZT
reversetranscriptase inhibitor (anti-retroviral therapy)
Avery, McCloud, and McCarty
DNA isolated from heat killed virulent bac turns live non-virulent bac to encapsulated virulent bacteria
hershey-chase
radioactive labeled DNA or coat infecting bacteria
chargaff’s rule
%G=%C and %A=%T but he ratios of the different pairs can be different
DNA backbone
deoxyribose sugar backbone with phosphodiester bonds
grooves
major and minor; bases in the major groove are more accessible than in the minor groove
stacking energy
higher for more purine content (G-C stacked with G-C)
lower salt concentration (DNA effect)
less [salt] will decrease Tm because there is less cations to nutralize exposeed phosphate neg charges
pH (DNA effect)
high pH (melts DNA but leaves phosphodiester bonds intact); low pH hydrolyzes phosphodiester bonds
increased chain length (DNA Tm)
longer chains have higher Tm
complementary sequences
good way to distinguish DNA mismatches
5-methylcytosine
has consequences in gene regulation and mutagenesis
deamination of nuc bases
can tun 5-MeCytosine into thymine, guanine into xanthine etc; nitrous acid or precursors can speed up this process
depurination of deoxyribose by hydrolosis
leads to breakdown of phosphate backbone
UV cross linking of DNA
thymin 2+2 rxn, leads to DNA kinks
Hydroxyl radicals
can add hydroxyl groups to DNA bases
alkylating agents
nucluophilic bases can get alkylated
intercalating agents
disrupt base stacking screw up DNA structure; eg. actinomycin D or doxorubicin
supercoiled DNA
+supercoiled DNA = like a knotted phone cord, -supercoil=like stretched cord
topoisomerases
relaxes supercoils to normal DNA form which is necessary for DNA replication. Drugs inhibit topoisomerase to prevent cancer cells from raplicating
nucleoside analogues do what?
mimic chemistry of natural nucleosides except they typically block transcription. Useful for antiviral therapies
RNA vs DNA
RNA turns over faster, much more susceptible to hydrolysis (can hydrolyze itself), can have implications in gene expression; no double helix so can have different conformations and also act as enzymes
puromycin
nucleotide analogue that binds to the 3’ end of tRNA and blocks translation
rRNA
the business end od the small and large subunits (proteins hang off the RNA scaffold)
3 classes of RNA
structural RNA; Regulatory RNA, Information containing RNA
Structural RNA
rRNA, tRNA, small nuclear RNA, small nucleolar RNA
Regulatory RNA
microRNA, small interfering RNA
Information containing RNA
mRNA
transcription direction
unidirectional and processive, 3’ -OH group on the growing nucleophilically attacks the proximal phosphate of a new NTP molecule
RNA transcription initiation
RNA pol binds to promoter sequence; pol melts DNA near transcription start site; Pol catalyzes first phosphodiester linkage between first two NTPs
RNA transcription elongation
Polymerase advances from 3’ to 5’ down template DNA trand making the new 5’ to 3’ RNA
RNA transcription termination
at transcription stop site, pol releases and the completed RNA releases and dissociates from DNA
E.Coli RNA pol
makes mRNA, tRNA, and rRNA
RNA pol I
makes rRNA
RNA pol II
makes mRNA, snRNA, miRNA and lncRNA
RNA Pol III
makes tRNA
RNA pol promoters
promotor proximal elements, TATA box
TATA box
at -30, TATA binding protein clamps onto DNA minor groove and directs assembly of the pre-initiation complex
xeroderma pigmentosum
can be caused by a defect in TFIIH which functions in transcription and DNA repair
5’ and 3’ UTR
parts of exons upstream or downstream of the ATG start site or the termination site
alpha-amanitin
competitive inhibitor of pol II, blocks chain elongation by preventing translocation
rifampicin
binds bacterial RNA pol and blocks the RNA exit channel
3 ways pre mRNA’s are processed
capping, splicing, cleavage/polyadenylation
mRNA Capping
happens on 5’ end: 1) cleave triphosphate (triphosphatase), 2)add a GMP (gyanylyltransferase) and then methylate the 7 position of the guanine (guanine-7-methyltransferase). Ultimately, the 5’ end will have a triphosphate linkage to the 5’ carbon of 7’methyl guanosine
5’ cap function
protects from degradation by 5’ exonucleases; cap binding protein regulates nuclear exportation. CBC is replaced by eIF-4E in the cytoplasm
overexpression of eIF4E
malignant transformation
5’ splice site
GU
3’ splice site
AG
initiation codon
AUG (codes for Met)
termination codon
UAG, UAA, UGA
splicing
2’ OH of branchpoint attacks 3’ phosphate on the 5’ end, kicking off 3’ -OH. That 3’ -OH attacks the 5’ phosphate on the 3’ end. Lariat intron gets excised
poly A consensus sequence
AAUAAA
polyadenylation rxns
after the AAUAAA endonuclease cleaves subsequent nucleotides; then polyadenylate polymerase adds a bunch of adenosines
thallasemia
defect in humoglobin production; caused by mutations in poly A consensus sequence
alternative splicing
allows many different proteins to be encoded by a single gene
U1 snRNA
recognizes the 5’ splice site
U2 snRNA
recognizes the branch point
genetic disorders caused by splicing defects
marfan syndrome disrupted splicing of the fibrilin gene
AAUAAA and termination
when pol II reaches AAUAAA termination of transcription is signalled
poly A tail function
protection, stabilization, enhanced translation
example of different proteins from alternative polyA site choice
secreted form mRNA plasma cells and membrane form mRNA cells
altered PolyA site and cancer
shortening of the 3’ UTR can activate cancer oncogenes
Bidirectional DNA replication
replication begins from a site of origin and proceeds in both directions from there
sites of origin
prokaryotes have one site on each chromosome; eukaryotes have multiple sites (humans have 100’s); usually multiple short repeats
semiconservative DNA replicaiton
each new DNA strand has a parent and a daughter strand
DNA synthesis
unidirectional polarity: proceeds in the 5’ to 3’ direction, is semi discontinuous.
replication forks
site at which DNA synthesis occurs,
origin binding proteins
recognize and bind origins of replication (tend to be A-T rich since they ar easier to melt; multiple short repeats too)
helicases
separate and unwind DNA parent strands
single strand binding proteins
bind to the single (parent) strands and prevents re-annealing to allow for stability and synthesis
topoisomerases
relaxes supercoiling to relieve torsional stress ahead of the replication fork
DNA Gyrase
a topoisomerase inhibited by quinolones, found mostly in prokaryotes
RNA primer
DNA polymerase canno initiate de novo synthesis: needs RNA primer (~10 nucleotides)
primase
catalyzes the reaction for synthesis of RNA primer
DNA pol III
elogation of the new chain, it is the major replicative enzyme, has sliding clamp
sliding clamp
part of DNA pol III; keeps the pol attached over a long distance, giving pol III a high processivity
DNA pol I
removes RNA primer and copies it into DNA
Eukaryotic DNA replication:
used pola, pold, and pole and is similar to prokaryotic. pola, serves as primase tho
okazaki fragments
discontinuous fragments of the lagging strand, joined together by DNA ligase
DNA ligase
forms phosphodiester bonds between okazaki fragment “nicks”
5’-3’ exonuclease
removes RNA primer in eukaryotes (done by Pol I in prokaryotes)
fidelity of replication
overall error rate of 10-9 to 10-10
polymerase fidelity
hydrogen bonds and geometry of complementary base pairs allows for 1 error per 10k to 100k nucleotides
proofreading
performed by 3’to5’ exonuclease increases fidelity by 100 to 1000x
3’ to 5’ exonuclease
proofreading enzymes closely associated with polymerase complex, increased fidelity by 100-1000x; both polI and polIII have 3’to5’ exonuclease activity
post replicational repair processes
eg. mismatch repair; further increase fidelity of repair
reverse transcription
synthesis of DNA from an RNA template; catalyzed by reverse transcriptase (commonly in retroviruses), telomerases also have reverse transcriptase activity
