cell bio proteins and enzymes

DNA polymerase

key enzyme in DNA replication

• all DNA polymerases add deoxyribonucleoside 5'-triphosphates to the 3' hydroxyl group of a primer strand.

DNA polymerase I

polymerase found in prokaryotes that has 5’ to 3’ exonuclease activity to remove RNA primers from DNA strand. it also acts as a DNA polymerase and fills in gaps between lagging strands

what is special about DNA pol I

its one protein that does 2 job in lagging strand of prokaryotes

DNA polymerase III

main synthesizing DNA polymerase in leading and lagging strands in prokaryotes

DNA polymerase α

relatively slow DNA polymerase that work with primase to make RNA primer in lagging strand of eukaryotes.

DNA polymerase ε

DNA polymerase that does synthesis in leading strand in eukaryotes

DNA polymerase δ

5’ to 3’ DNA polymerase that synthesizes DNA in eukaryotes lagging strand

• also fills in gap between Okazaki fragments

primase

RNA polymerase that is used in DNA replication to generate primers to initiate synthesis of Okazaki fragments

• RNA pol can generate polynucleotides de novo (from scratch) w/o pre-existing primer

DNA ligase

enzyme that joins DNA fragments together

RNase H

protein that is a 5’ to 3’ exonuclease used in lagging strands to remove RNA primers in eukaryotes

what are the processive DNA polymerases in both prokaryotes and eukaryotes?

• DNA pol III

• DNA pol δ

• DNA pol ε

helicase

enzyme that separated and stabilizes dsDNA into ssDNA - it catalyzes the unwinding of DNA

• needs ATP to break hydrogen bonds

• works with SSB

single stranded DNA binding proteins (SSB)

keep separated ssDNA strands apart till DNA polymerase can do its job

• works with helicase

sliding clamp protein

holds DNA pol in place so it can go faster (looks like a donut)

• naturally closed protein, requires help from other protein to open up and wrap around DNA

• known as PCNA in eukaryotes

clamp loading protein

uses ATP to open up the sliding clamp protein so its no longer in its closed form

• RFC in eukaryotes

topoisomerase (I & II)

enzyme that cuts supercoiled DNA strands, and once DNA unwinds (transient break) and no knots are present, enzyme joins strands back together

what is special about topoisomerase

its a type of nuclease and ligase

initiator

protein that recognizes origin of replication for DNA replication in prokaryotes and starts recruiting other protein such as primase and helicase

ORC

origin recognition complex - protein complex that initiates DNA replication at eukaryotic origins

• recognizes and binds to origin of replication and recruits other proteins

telomerase

enzyme that make telomeres through reverse transcriptase

• has its own RNA template

• part protein and part RNA

what mechanisms do processive DNA pol have to help ensure accurate DNA replications?

have proofreading activity that helps reduce error rate.

• exhibit 3’ to 5’ exonuclease activity

photolyase

light induced enzyme in plants that help repair thymine dimer

• not in humans

MutS

protein that recognizes mismatch in prokaryotes during mismatch repair

• work with MutL to do direct excision

MutH

protein that cleaves the strand with the mismatch adjacent to methyl group on parent strand

• in prokaryotes during mismatch replair

MutL

works with MutS to directly excisise strand of DNA with mismatch during mismatch repair

• in prokaryotes

MSH

mutS homolog - recognizes mismatch in eukaryotes

MLH

mutL homolog- excises nucleotides on DNA strand with the mismatch till it encounters a single stranded break

• part of mismatch repair in eukaryotes

DNA glycosylase

enzyme that removes nucleotide base U, leaving behind phosphate and sugar, creating an AP Site

• part of deamination base-excision repair

AP endonuclease

enzyme that cleaves DNA at an AP site

• part of deamination base-excision repair

deoxyribosephosphodiesterase

enzyme the breaks phosphodiester bonds

• part of deamination base-excision repair

excinuclease

complex of proteins that remove thymine dimer and additional nucleotides around it

• parts of nucleotides excision repair of thymine dimers in humans

Ku70-Ku80

proteins that recognize DSBs and recruit other proteins to fix it

• part of DSB repair by NHEJ

DNA-PKcs

DNA dependents protein kinase, add phosphates and changes activity of other proteins

• part of DSB repair by NHEJ

artemis

a nuclease that is part of DSB repair by NHEJ

Rad51

protein that help in repairing DNA DSB - recognizes overhangs and matching it to undamaged sister chromatid during G2 phase

• part of DSB repair by Homology-Directed Repair

Rag 1 & Rag 2

recombinase enzymes in antibodies that create more diversity by splicing certain pieces of DNA

• these enzymes recognize recombination signals (RS) which are contained within coding sequences (different V, D regions)

AID

activation induced deaminase - enzyme that deaminates cytosine into uracil which leads to base excision repair

• plays a key role in class switch recombination and somatic hypermutations

RNA polymerase

key enzyme in transcription - transcribes in 5’ to 3’ direction

describe RNA polymerase in bacteria

has 5 subunits: α (2 copies), β , β’ , ω, σ

describe the core polymerase in prokaryotes

includes the 4 subunits: α (2 copies), β , β’ , ω

• responsible for polymerase activity

describe the role of σ in transcription

it is a subunit that encounters the promoter sequences, forming a closed promoter complex and initiation transcription

• once transcription is initiated σ is released

what are the different RNA polymerase in eukaryotes

RNA pol I, RNA pol II, RNA pol III, mitochondrial RNA pol

RNA pol II

transcribes all protein coding genes and requires initiation fact that are not associated with the polymerase

• synthesizes mRNA, miRNA, lncRNA

what is RNA pol II recruited to promoter by?

general transcription factors - TFIID, TFIIB, TFIIF, TFIIE, TFIIH

describe TFIID - what are the 2 subunits of it and what does each do?

recognizes TATAA sequences (TATAA box)

• TBP (tata binding proteins): binds to TATAA box

• TAF (TBP associated factor): binds to other promoter elements

describe TFIIB

links TBP and RNA pol II, positioning it at TSS

• (TBP part of TFIID)

describe TFIIF

stabilizes RNA pol II to the complex and its binding to the promoter.

• (kind of like a double checking mechanism that ensures stability and correct positioning)

what are TFIIB and TFIIF critical for?

recruitment of RNA pol II

describe TFIIE

plays a preparatory role in transcription initiation - primary function is to help get everything ready for the actual start of transcription

• stabilizes interactions between complex

• recruits TFIIH

describe TFIIH

TF that acts as a helicase and a CTD Kinase

• unwinds DNA at TSS, allowing access to RNA pol II

• phosphorylates the carboxy-terminal domain of RNA pol II which initiates transcription

mediator

protein that facilitlates interaction between RNA pol II and regulatory factors - holds GTFs and RNA pol II in place

• once transcription begins mediator gets left behind

RNA endonucleases

cleave pre-mRNA at polyadenylation signal causing the rest to be degraded by an exonuclease

RNA pol I

RNA polymerase that transcribes for ¾ units of rRNA (18S, 5.8S, 28S)

• multiple RNA pol I can transcribe the same rRNA gene simultaneously.

• crucial for the efficiency , bc ribosomes are needed in large quantities

RNA pol III

RNA polymerase that transcribes for ¼ units of rRNA (5S) and tRNA

what is a unique thing about about RNA pol III

its transcription factors are downstream of the polymerase

• usually upstream of other polymerases

snoRNP

a type of protein complex known as small nucleolar ribonucleoprotein.

• important with ribosome synthesis - hence the nucleolar, which refers to the nucleolus where ribosomal transcription takes place.

methyl transferase

enzyme that methylates ribose attached to specific nucleotide bases during ribose methylation, a type of base modification in rRNA that contributes to stabilization.

• a snoRNP

uridine isomerase

enzyme that converts ribose uridine to ribose pseudouridine during uridine isomerization. conversion stabilizes the rRNA molecule by altering its immunogenicity.

• a snoRNP

Rnase P

a ribozyme (RNA with enzyme activity)

• responsible for cleaving the 5' leader sequence from pre-tRNA

• essential for the maturation of functional tRNA molecules

guanylyl transferase

enzyme that participates in 5’ end capping - putting nucleotides backwards to protect mRNA from exonuclease and prevent it from being degraded.

poly A polymerase

enzyme that add a poly A tail at the 3’ end of mRNA. regulates transcription rates and stability

• directed by 3 sequence elements, one upstream of poly A site, one downstream of poly A site and one in transcribed region

snRNP

small nuclear ribonucleoprotein particles

• particles bc contains both RNA and proteins

• crucial during splicing of introns in mRNA

spliceosomees

• a large RNA-protein complex composed of snRNAs (U1, U2, U4, U5, U6) and proteins.

• facilitates splicing of pre-mRNA by removing introns and ligating exons, essential for producing mature mRNA.

describe U1, U2, U4, U5, U6

• U1 recognizes 5’ splice site

• U2 recognizes branch points

• U4, U5, U6 recognize 3’ splice site

what makes RNA capable of participating in processes, such as splicing

RNA can do things unlike DNA. so its capable of splicing and ligating

• U2 and U6 can catalyze the reaction in the absence of protein

splicing factors

proteins that facilitate splicing by guiding snRNPs to the correct splice sites

β - galactosidase

enzyme that breaks down lactose into glucose

CAP

a regulatory protein that enhances the transcription of certain genes in response to low glucose levels.

• for it to be activated it needs to be bound to cAMP

cohesin

proteins that act like glue and stick DNA together

• crucial in DNA looping

CTCF

an architectural protein involved in regulating the organization of chromatin

• A molecule that dimerizes and recognizes specific DNA sequences, helping to organize DNA within defined regions known as topologically associating domains (TADs).

NELF & DELF

negative regulatory factors that instruct RNA polymerase II to pause after transcription initiation, once the first exon has been transcribed.

P-TEFb

displace NELF protein from RNA pol II and phosphorylates CTD at a new site, allowing the recruitment of spliceosomes

• allows for elongations of RNA pol II

HAT

histone acetyltransferases - enzymes that transfer acetyl groups to histones, resulting in a more relaxed DNA-histone interaction and generally promoting gene activation.

• a coactivator

HDAC

histone deacetylases - enzymes that remove acetyl groups from histones, leading to tighter DNA-histone interactions and generally resulting in gene repression

• a corepressor

DNMT

DNA methyltransferase - enzyme that add methyl groups to DNA nucleotides

PRC2

polycomb repressive complex 2 - recognizes trimethylated histone tails (signature of promoter) and recruits other proteins to trimethylate neighboring histones

• relates to epigenetic modification

UHRF1

protein complex that recognizes hemimethylated cytosine and recruits DNMT to add a methyl group to newly synthesized DNA strand

TET

proteins that play a role in DNA demethylation