Biochem Exam 4

NTP to dNTP how, what’s used

by ribonucleotide reductase, NADPH used and water released

how ribonucleotide reductase is regenerated

through electron transport chain

electrons from NADPH to either glutaredoxin or thioredoxin then to ribonucleotide reductase

structure o ribonucleotide reductase

2 alpha and 2 beta subunits

2 -SH on each alpha, Tyr radical stabilized by Fe in beta, 2 regulatory sites on each alpha

primary regulation site on ribonucleotide reductase

ATP activates, dATP inhibits

substrate specificity site on ribonucleotide reductase

high ATP and dATP → less specificity for adenine, more specificity for UDP, CDP, etc

mechanism for ribonucleotide reductase reaction

Tyr radical takes H from 3’ → OH on 2’ protonated by -SH → H2O leaves 2’ → other -SH gives H to 2’ → 3’ takes back H and Tyr radical regenerated

how ribonucleotide reductase is inactivated by dATP

oligomerization, radical forming path is disrupted bc residues are exposed to solvent

how dTMP is made

CDP or UDP to dCDP or dUDP by ribonucleotide reductase → dCDP or dUDP to dCTP or dUTP by nucleoside diphosphate kinase → dCTP to dUTP by deaminase → dUTP to dUMP by dUTPase → dUMP to dTMP by thymidylate synthase

how thymidylate synthase works

by turning N5,N10-methylene-tetrahydrofolate into 7,8-dihydrofolate

regenerate via tetrahydrofolate

medical application of thymidylate synthase, ex

inhibiting it stops DNA replication but not RNA so target for cancer

fluorouracil inhibits thymidylate synthase directly, methotrexate inhibits regeneration via tetrahydrofolate

how purines are degraded

AMP becomes hypoxanthine then xanthine, GMP becomes xanthine, both become uric acid and excreted

how pyrimidines are degraded

nitrogen parts become urea and excreted, carbon parts become succinyl-CoA and fed into TCA cycle

gout caused by, treatment

overproduction of uric acid

inhibit xanthine oxidase (xanthine to uric acid)

recycling of nucleotide bases

free adenine, hypoxanthine, guanine combine with PRPP to become AMP, IMP, GMP

nucleotides linked by

phosphodiester bonds

phosphate on 5’ → OH on 3’

geometry of 5 carbon sugar, significance

puckered, 2’ and 3’ stick up and down

change distance between 5’ and 3’

charge, structure of backbone

negatively charged

phosphate and pentose

AT vs GC

AT has 2 hydrogen bonds, GC has 3

Chargaff’s Rule

A=T and G=C in all species but DNA composition differs

Griffith’s experiment

dead virulent type and alive non-virulent type both injected and killed mouse, established transforming principle

Avery/MacLoud/McCarty experiment

DNA not RNA or protein is responsible for transforming principle

Rosalind Franklin/Watson/Crick experiment

took x-ray image of DNA -> DNA is helical, found base pairs per turn, size of pitch, strands aren’t symmetrical, diameter of DNA

major vs minor groove

major is wide and shallow, minor is narrow and deep

DNA stabilized by

hydrogen bonds and stacking interactions

3 forms of DNA

B form - right handed, most stable, forms spontaneously

A form - right handed, tight and more bases per turn

Z form - left handed, rare

DNA denatured by, easier where

heat or pH

AT-rich sequences denature easier and faster

transposon is, causes what

genetic elements that can jump to different locations within genome

cause mutations that lead to diseases and aging

different RNA structures

single stranded, hairpin double helix, internal loop

how DNA is recognized

phosphate backbone, sequences specific (mostly at major groove), G quadruplexes (tertiary structure especially at telomerase), amino acids that form hydrogen bonds with bases

advantage of major groove

can discriminate A, T, G, C

only AT vs GC discriminated in minor groove

helix turn helix and homeodomain

recognition alpha helix positioned in major groove

homeodomain is larger

zinc finger

coordinated Zn ions, bind super tightly to DNA

leucine zipper, basic helix loop helix

2 helix stradle around DNA, held together by zipper region (Leu every 7th)

basic helix loop helix has 2 sets of domains, have a loop

purpose of supercoiling

compacts DNA, relieves strain

DNA is underwound - meaning, why

strained and fewer helical turns than expected, stabilized by supercoiling, makes strand separation easier

what is Lk, how it changes

number of times one strand wraps around another, # of bp / bp per turn

underwinding decreases Lk while overwinding increases it, increase is positive supercoiling while decrease is negative supercoiling, by topoisomerase

type 1 topoisomerase

change Lk by 1, cleaves one strand of DNA

how topoisomerase I works

Tyr binds DNA and cleaves one strand -> passes other strand through break -> Tyr religates broken strand

type 2 topoisomerase

change Lk by 2, cleaves both strands, uses ATP

how topoisomerase II works

enzyme binds 2 DNA -> 1 DNA cleaved -> second DNA passed through break, 2 ATP bound -> broken DNA relegated and other DNA is released -> 2 ADP released

2 types of supercoiling

plectonemic (less compact, in bacteria), solenoidal (more compact, in eukaryotes)

structure and purpose of histones

made of 8 subunits (2 each of H2A, H2B, H3, H4), positively charged

packages DNA into nucleosomes

wrapping DNA around histone requires

topoisomerase, negative supercoiling (Lk decreases by 1)

nucleosomes organized into, how

chromosomes

by cohesins and condensins

chromatin modification, purpose

modifications made on histone tails

regulate chromatin structure and chromatin-based processes

2 types of chromatin assembly factors

histone chaperones - no ATP, shuttle histones to nucleus, help assembly/disassembly/exchange of histones

ATP-dependent remodelers - slide or eject nucleosomes, composition changes during development so guides developmental decisions

Meselson-Stahl experiment

grown in 15N -> switched to 14N and divide once -> all DNA were hybrid -> divide in 14N again -> some hybrid, some only 14N

confirmed DNA replication is semi-conservative

DNA polymerase requires

template strand, primer with 3’ OH to extend, dNTP, Mg2+ (coordinated to Asp in polymerase, holds onto phosphate to make phosphodiester bond)

accuracy of DNA polymerase, exception

active site can only accommodate correct dNTP

Y family polymerases have more open active site

types of DNA polymerase

I - 5'→3’ and 3’→5’ exonuclease activity

II - 3’→5’ exonuclease activity

III - 3’→5’ exonuclease activity, high polymerization rate and processivitiy

Y family (IV, V, more error prone)

10 proteins required for DNA replication initiation

DnaA (opens up DNA at oriC sequence)

DnaB (helicase, unwinds DNA)

DnaC (required for DnaB

HU (histone like)

FIS and IHF (both binds to DNA)

primase (makes RNA primers)

SSB (binds and stabilizes single stranded DNA)

DNA gyrase (topoisomerase II)

Dam methylase (methylates sequences at oriC)

purpose of DNA polymerase I, what happens after

removes RNA primers (5’→3’ exonuclease) and adds dNTP, DNA ligase uses ATP to seal the nick

how DNA ligase works

adenylylation of enzyme which becomes enzyme-AMP → activates 5’ phosphate to become just enzyme → nucleophilic attack by 3’ OH

6 proteins required for DNA replication elongation

SSB (binds and stabilizes single stranded DNA)

DnaB (helicase, unwinds DNA

primase (makes RNA primers)

DNA polymerase III (elongates new strands)

DNA polymerase I (gets rid of primers and fills in gaps)

DNA ligase

structure of DNA polymerase III, how it loads

clamp loader with 3 subunits (each with pol III core, beta sliding clamp)

clamp loader has 3 ATP and binds DNA -> becomes ADP and leaves -> beta clamp stays on

DNA polymerase III for lagging strand

new beta clamp loaded and old beta clamp is left behind for each Okazaki fragment

trombone model (lagging strand loops, 2 subunits on at a time)

issue with DNA replication termination in bacteria, solution

lead to interlocked catenated chromosome

topoisomerase IV (type 2) separates them

issue with DNA replication in eukaryote, solution

rate slower and chromosomes longer

multiple origins of replication

2 steps for initiation of eukaryotic DNA replication

1\. form pre-RC (CDC6 and ATP binds to ORC, helicase bound by CDT1)

2\. coordinate activation (CDC6 and CDT1 off, ATP on polymerase becomes ADP and separates from helicase)

purpose of cyclin

to ensure origin only fires once per cell cycle

how telomeres are made

telomerase has RNA template within, uses it to extend 3’ end → RNA primase makes primer at end → DNA polymerase, DNA ligase → RNA primer removed by RNAse → single stranded part protected by telomere-binding proteins

ex of deamination

occur frequently - cytosine to uracil, methylcytosine to thymine

more rare - adenine to hypoxanthine, guanine to xanthine

deamination rate increased by

deaminating agents (nitrous acid precursors)

UV irradiation leads to

bonding between adjacent thymines, distort geometry of double helix and blocks replication, lead to xeroderma pigmentosum

depurination

hydrolysis of guanosine → guanine leaves, apurinic residue remains

alkylation

methylation of guanine creates methylguanine, can’t pair with cytosine and pairs with thymine -> mistake replicated and a strand forms with AT

4 mechanisms for DNA repair

mismatch repair, base excision repair, nucleotide excision repair, direct repair

mismatch repair takes advantage of

parental strand is methylated but it takes few minutes for new strand to also become methylated

mechanism for mismatch repair

MutL-MutS binds to mismatch -> MuH binds and finds methylated site -> MutH cleaves unmethylated strand -> exonuclease activity degrades DNA from methylated site past the mismatch -> DNA polymerase III fills in the strand

base excision repair

base specific glycosylate cleaves just the base → AP endonuclease removes sugar → DNA polymerase I fills in the right nucleotide -> DNA ligase

nucleotide excision repair

used to remove bulky lesions, excinuclease makes 2 cuts -> filled in with DNA polymerase and ligase

direct repair

methylguanine back to guanine using methyltransferase, methyltransferase is suicide enzyme (can’t be regenerated) so expensive

3 types of RNA polymerase for eukaryote

I for rRNA; II for mRNA, snRNA, miRNA; III for tRNA and 5S rRNA

how RNA polymerase differs from DNA polymerase

doesn’t need primer, no exonuclease activity (for the most part)

RNA strand code compared to DNA strands

RNA complementary to DNA template strand, same sequence as nontemplate / coding strand

how RNA polymerase works

separates and puts back DNA strands within (isoenergetic), NTP fed in a channel and RNA exits out other channel, unwinding in front and rewinding behind to deal with strain

Transcription initiation in e coli at, what’s special about it

promoter

\-10 and -35 especially are very conserved, polymerase binds tightest when most similar to consensus sequence

Transcription in e coli

RNA polymerase binds to sigma70 -> binds to promoter, transcription initiated -> sigma70 dissociates, NusA attached -> elongation -> NusA and RNA removed when terminated

clamp of RNA polymerase, significance

clamp open during initiation but closed during elongation, closed means processive so RNA polymerase doesn’t fall off

rho independent termination

weak base pairs (AT) facilitates melting of duplex, stem loop hairpin pries polymerase off

rho dependent termination

rho helicase binds to rut site -> uses ATP to migrate along mRNA to polymerase -> rho helicase separates mRNA from DNA template

RNA polymerase II has, significance

CTD

post-translational modification marks deposited, phosphorylated during initiation then dephosphorylated in termination

2 of transcription factors needed by RNA polymerase II

TBP (recognizes and binds TATA box)

TFIIH (unwinds DNA, phosphorylates CTD, recruits nucleotide excision repair proteins)

synthesis of RNA and DNA from RNA, significance

RNA to RNA by replicase, COVID

RNA to DNA by reverse transcriptase, HIV

how reverse transcription works

RNA to RNA-DNA hybrid by DNA polymerase that’s primed by tRNA -> single stranded DNA by RNase H -> double stranded DNA by DNA polymerase that’s primed by 3’

post transcriptional processing in eukaryotes

5’ cap, splicing, poly A tail

5’ cap

7-methylguanosine attached to 5’ end via 5’,5’-triphosphate linkage, sometimes last few nucleotides are methylated at 2’ OH

how poly A tail formed

polyadenylation factors recognize signal → endonuclease cleaves after signal, RNA polymerase leaves → polyadenylation factor leaves, polyadenylate polymerase adds A in non-templated way, uses ATP

group I splicing

OH on guanosine attacks phosphate at 5’ splice site and attaches -> same OH then attacks phosphate on 3’ splice site

significance of group I introns

ribozymes, traps guanosine to prevent from floating away

group II splicing

OH on adenosine within intro attacks 5’ splice site, forms lariat -> OH on 5’ splice site attacks phosphate on 3’ splice site -> lariat leaves

nuclear splicing

snRNPs bind (U1 to 5’ splice site, U2 to both branch point containing bulged A and 3’ splice site), ATP to check proper base pairing (proper bp necessary for ATP hydrolysis), other snRNP rearrange to facilitate chemistry (branch point attached to 5’ splice site -> lariat)

5’ cap formation and splicing occurs where

recruited by RNA polymerase and occurs on it

rRNA processing in prokaryote

pre-rRNA processed and cleaved into 16S rRNA, 23S rRNA, 5S rRNA, and tRNA

rRNA processing in eukaryote

pre-rRNA processed and cleaved into 40S and 60S then transported to cytoplasm

tRNA processing

cut by RNase P and RNase D, base modifications, splicing

tRNA splicing

endonuclease to cut out intron -> uses ATP to activate 3’ splice site then nucleophilic attack

start and stop codons

AUG (Met) is start, UAA/UAG/UGA are stop codons

degeneracy

3rd position of codon doesn’t matter, minimize effects of mutation

wobble pairing

I pairs with A, U, C

U binds to A and G

G binds to C and U

A only binds to U, C only binds to G