DNA & RNA Flashcards
Dinucleotide sequence marking the 5’ end of intron
GU
Dinucleotide sequence marking the 3; end of intron
AG
What is the spliceosome made of?
Small nuclear ribonuclear proteins (snRPS) and snRNAs U1, U2, U4, U5, U6
U1 snRNA
Contains a sequence complementary to the mRNA sequence near the 5’ splice site of introns
Relative solubility of DNA components
Nucleotide > Nucleosides > Base
Pyrimidines > Purines
Gout
A defect in hypoxanthine-guanine phosphoribosyl transferase; leads to the accumulation of purines (uric acid) in tissues, often presenting as red/swollen, most commonly in the big toe
Lesch-Nyhan syndrome
A genetic defect in hypoxanthine-guanine phophoribosyl transferase (HGPRT); leads to the accumulation of purines (uric acid) in tissues; presents as severe gout, kidney problems, cognitive defects, and self-injuring behavior
Chargaff’s Rule
The ratio of purines (G + A) and pyrimidines (T + C) must be equal as a result of G-C and A-T base pairing; %G = %C and %A = %T
Tm
Temperature at which a given DNA strand is 50% denatured; affected by salt concentration, pH, DNA chain length, and GC content
Deamination
Deamination of 5-methyl cytosine converts 5mC to thymine
Depurination
Spontaneous hydrolysis of a purine base from it’s sugar-phosphate backbone, leaving the DNA backbone vulnerable to cleavage
Nucleoside analogue drugs
Inhibit DNA synthesis by mimicking normal nucleotide substrates in the DNApoly active site; ex: AZT inhibits HIV viral DNA synthesis by reverse transcriptase
Classes of RNA
Structural (rRNA, tRNA, snRNA, snoRNA)
Information-containing (mRNA)
Regulatory (miRNA, siRNA)
Puromycin
Antibiotic; inhibits bacterial translation by mimicking the 3’ tRNA acceptor region and interacting with the ribosome to cause premature peptide release
B form DNA
The most common form of DNA; two anti-parallel strands intertwined in a right-handed helix
DNA Methylation
Usually occurs on C within CpG dinucleotide sequences; performed by DNA methyltransferase; generally down regulates gene expression
Alkylating agents
React with nucleophilic groups on purine/pyramidine bases
RNA Polymerase I
Synthesizes pre-ribosomal RNA
RNA Polymerase II
synthesizes mRNA
RNA Polymerase III
synthesizes tRNA
TATA Box / Initiator sequence
DNA control element located 25-30 bp upstream from the transcription start site; bound by TATA-binding protein (TBP); this is the site at which general TFs bind
TFIID
Contains TATA-binding protein (TBP) as well as TBP-associated factors (TAFs)
a-amanitin
Death cap mushroom toxin; blocks the translocation of RNA polymerase II by interacting with its bridge helix
Rifampicin
Antibiotic; binds to RNA polymerase at the exit channel and sterically occludes the formation of mRNA chains > 2-3 nucleotides long
RNA Polymerase II pre-initiation complex
RNA Polymerase II
TFIID (TBP + TAFs)
TFIIA, TFIIB, TFIIE, TFIIF, TFIIH
TFIIF
Part of RNA polymerase II preinitiation complex; phosphorylates RNA polymerase II, which activates it
TFIIH
Part of RNA Polymerase II pre-initiation complex; CDK7 subunit phosphorylates Pol II CTD; has some helicase activity, and functions in nucleotide excision repair (NER)
Xeroderma Pigmentosum
Autosomal recessive disorder of DNA repair, usually caused by mutation in one of the nucleotide excision repair pathways - often TFIIH
5’ splice site consensus sequence
GU
3’ splice site consensus sequence
AG
PolyA tail cleavage site consensus sequence
AAUAAA 10 to 30 nucleotides upstream from the cleavage site
U2 snRNA
Base pairs with the intron at a position corresponding with the branch point A residue, causing a local distortion in the primary transcript that activates the branch point A
DNA Polymerase III
The main polymerase involved in DNA replication; Pol III “holds on” to the DNA via a sliding clamp protein and has 3-5’ exonuclease activity that enables proofreading
DNA Polymerase I
Mostly involved in replacing RNA primer with DNA; has exonuclease activity in the 5-3’ direction that removes the RNA primer
Hereditary non-polyposis colorectal cancer (HNPCC) / Lynch Syndrome
Caused by mutations in the machinery that performs mismatch DNA repair
Cockayne Syndrome (CS), Xeroderma Pigmentosum, and Trichiothiodystrophy
Caused by mutations in genes that mediate nucleotide excision repair (NER), often TFIIH
Deamination of C yields…
U; can result in permanent mutation of a CG base pair to a TA base pair
DNA Mismatch repair
Fixes errors in nucleotide incorporation made by DNA polymerase during DNA replication
MutS and MutL (proks) / MSH and LH (euks)
Proteins that recognize mismatched DNA nucleotides as part of DNA mismatch repair pathway; these proteins recognize the error on the newly synthesized strand, which is unmethylated
Nucleotide Excision Repair (NER)
Removes DNA lesions that distort the DNA structure and therefore block RNA or DNA polymerase (i.e. thymine dimers) - global and transcription-coupled pathways
Base excision repair (BER)
Removes DNA lesions that do not cause distortions that block polymerase function, and therefore may be missed by NER; glycosylase enzymes hydrolyze the N-glycosidic bond to remove the damaged base, producing an AP site which is then removed by an AP-specific endonuclease
Homologous Recombination (HR)
Repairs double-stranded DNA breaks between homologous ends
Non-homologous end joining (NHEJ)
Repairs double-stranded DNA breaks; does not require sequence homology between the two broken ends - may lead to insertion or deletion of nucleotides at the break point
Lesion Bypass Repair
Allows cells to continue replicating despite extensive DNA damage that cannot be repaired via other mechanisms; a DNA polymerase lacking 3-5; proofreading function is used to replicate past the lesion and is therefore highly mutagenic
DNA control element
Regulatory sequences of DNA that act locally by binding transcription factors or other regulatory proteins to control expression of the gene
B-Thalassemia
Inherited anemia associated with a mutation in the B-globin promoter, reducing the amount of B-globin mRNA that is transcribed
Hemophilia B Leyden
X-linked disorder caused by an inherited mutation of the promoter of the Factor IX gene; affected males produce only 1% of the normal amount of Factor IX clotting protein until puberty, when trx increases to 60% of normal
Fragile X Syndrome
Affected males have extra copies of a CGG trinucleotide repeat in the 5’ region of the Fragile X mental retardation 1 (FMR1) gene on the X chromosome, resulting in a failure to express the fragile X mental retardation protein (FMRP)
Sequence-specific DNA binding proteins
Trans-acting activators or repressors that bind directly to DNA control elements in their target genes to regulate transcription
Co-factors
Trans-acting activators or repressors that bind to sequence-specific DNA binding proteins to affect transcription
DNA Binding Domains - 4 categories
Helix-Turn-Helix
Zinc Finger
Basic Leucine Zipper Proteins (bZLP)
Basic Helix-Loop-Helix motif (bHLH)
Androgen Insensitivity Syndrome
Caused by a mutation in the androgen receptor (a zinc finger DNA binding protein), making them less responsive to androgens
Waardenburg Syndrome Type II
Caused by a mutation in the MITF gene, which encodes a bHLH DNA binding protein that plays a role in the development of melanocytes
Histone core complex
Octamer consisting of dimers of: H2A, H2B, H3, and H4
Histone H1
Bound to the linker DNA between histone octamers; functions to “slide” histones along DNA
Two classes of chromatin remodeling factors
ATP-dependent
Reversible modifiers
SWI/SNF family
ATP-dependent chromatin remodelers
Histone Acetyltransferaces (HATs)
Class of reversible chromatin modifiers; acetylate the N-termini of histones, neutralizing the positive charge on it’s N-terminal lysine AA and “freeing” the histone tails from electrostatic reactions with the DNA phosphate backbone
Rubinstein-Taybi Syndrome
Results from mutations in one copy (haploinsufficiency) of the CREB binding protein (CBP) gene; CBP is a transcriptional co-activator for many TFs and is a histone acetyltransferase; presents as growth regardation, mental retardation, craniofacial dysmorphism
Reversible modification of chromatin
Activators recruit histone acetyl transferaces (HATs) = activator-directed hyperacetylation
Repressors recruit histone de-acetyltransferaces (HDACs) = repressor-directed histone deacetylation
Tamoxifen
Tamoxifen antagonizes estrogen by binding to the estrogen receptor (ER) and preventing recruitment of HAT co-factors (activators)
NFKB pathway
NFKB protein is usually held in the cytoplasm by binding to its inhibitor (IKB); phosphorylation of IKB targets it for degradation, releasing NFKB to move into the nucleus where it turns on its target genes, including genes involved in inflammation
How does aspirin work?
In part by inhibiting the phosphorylation of IKB, keeping it bound to NFKB and preventing translocation of NFKB from the cytoplasm to the nucleus
Id family of proteins
Inhibitors of DNA-binding proteins (Id) heterodimerize with bHLH transcription factors to inhibit DNA binding - they contain the HLH-dimerization domain but lack the basic DNA-binding domain
Ribosomal subunits (prok & euk)
Prok - 30S & 50S (70S total)
Euk - 40S & 60S (80S total)
Stop codons
UAA
UAG
UGA
Shine-Delgarno sequence
Prokaryotic ribosomal binding site; essentially corresponds with initiator AUG codon
Initiation Factor 4E (IF4E)
Binds the 7-methyl guanosine cap on the 5’ unit of the mRNA; facilitates scanning of the ribosome down the primary transcript to the start AUG codon
Elongation factors (prok & euk)
Ef-Tu (in bacteria) & EF1A in eukaryotes; deliver charged aa-tRNA to the A site of the ribosome, burning GTP when it dissociates from a correctly matched codon/anti-codon pair
How many high energy bonds burned per AA?
4!
2 to charge aa-tRNA
1 to deliver aa-tRNA to A site
1 for translocation of ribosome
Initiation of Translation - Bacteria
IF1 and IF 3 bind the small subunit, which binds the mRNA at the AUG start codon via base pairing between the Shine-Delgarno sequence and the 3’ rRNA
IF2 (bound to GTP) delivers initiation Met-tRNA to the P site to pair with the AUG codon
GTP hydrolysis on IF2 release all IFs and signals binding of the large subunit
Rapamycin
An immunosuppressant drug; Inhibits mTOR (mammalian target of Rapamycin), which is a kinase that phosphorylates 4E-BP; inhibition of mTOR leads to dephosphorylation of 4E-BP, which is the state in which it binds eIF4E, silencing translation
Interferon
Produced in response to cellular infection, interferon signals other cells by activating a kinase that phosphorylates eIF2; phosphorylation of eIF2 inhibits it from facilitating the binding of initiation Met-tRNA to the ribosome thereby shutting down translation
Transferrin Receptor regulation
Brings iron into the cell; under conditions of low iron, IRE-BP is not bound to iron; it is free to bind the IRE element in the 3’ UTR of the transferrin receptor mRNA, protecting it from degradation and increasing translation of transferrin receptor
Under conditions of high iron, Iron is bound to IRE-BP, IRE-BP cannot bind the IRE element, and the mRNA is degraded
Ferritin Pathway
Binds excess levels of iron; mRNA has IRE element in it’s 5’ UTR
Under conditions of high iron, IRE-BP is not bound by iron; it binds the IRE and prevents ribosomes from accessing the AUG start codon, silencing translation
Under conditions of high iron, the IRE-BP is bound by iron; it cannot bind the IRE and ferritin is translated to bind excess iron
DNA Polymerase delta
Performs DNA synthesis on the lagging strand in eukaryotes; contains 3-5; exonuclease activity
DNA Polymerase alpha
Functions in the synthesis of the first ~20 DNA nucleotides immediately following the RNA primers; also synthesizes the first basepairs of each Okazaki fragment
DNA Polymerase epsilon
Performs DNA synthesis on the leading strand in eukaryotes; contains 3-5’ exonuclease activity
Z-DNA
Left Handed Helix form
Release Factor (RF)
Enters the A site (bound to GTP) in conjunction with a stop codon; hydrolysis of GTP induces release of the ribosomal subunits
