Quiz 3 Flashcards
flow of genetic information (basic)
protein coding gene transcribed by RNA polymerase into Pre-mRNA
Pre-mRNA processed to mRNA
mRNA is translated by ribosomes into protein
do all genes code for protein
no! some code for RNA only
what occurs in the nucleus
DNA to RNA transcript (transcriptional control)
RNA transcript to mRNA (RNA processing control)
what occurs in the cytosol
mRNA moves out of nucleus (RNA transport and localization control)
mRNA converted to inactive mRNA (mRNA degradation control) OR mRNA converted to protein (translation control)
THEN protein to active or inactive protein (protein activity control)
basic structure of nucleotide
base + ribose + phosphate
purines
9 membered - A or G
pyrimidines
C, T, or U - 6 membered
nucleoside
base + ribose
ribose vs deoxyribose
ribose has OH/H at 2 position, deoxyribose has H/H at 2 position
attachments in DNA
purine or pyrimidine is attached at 1 position on ribose, phosphate group at 5 position
how are nucleotides in nucleic acid chain linked
3’ hydroxyl to 5’ phosphate in a phosphodiester bond
how do you read a nucleotide chain
5’ to 3’
what stabilizes double stranded DNA
hydrogen bonding between G/C base pairs and A/T base pairs, base stacking, hydrophobic interactions.
what would cause DNA to have higher melting point
more GC
how do we get DNA to pack into the nucleus
using histones and spacer RNA `
nucleosome
DNA protein unit wrapped around histone
chromatin
DNA in protein bound form
heterochromatin vs euchromatin
heterochromatin is tightly wound and not available to transcription machinery. euchromatin is more loosely wound.
what is DNA packaging assisted by
polycationic amines (ex: Spermine)
how does DNA become available for transcription
acetylations, methylations, phosphorylations
what enzyme replicates DNA prior to cell division
DNA polymerase
lagging vs leading strand
leading strand is one on which replication fork is moving 3’ to 5’. Replication actually happening 5’ to 3’ because this is the only way DNA polymerase can work.
what transcribes RNA
RNA polymerase - only uses one strand of DNA, this is the “template strand”. Again, read in 3’ to 5’ but synthesized 5’ to 3’
coding strand
non template strand of DNA, has the same sequence as the coded RNA which was coded from the template strand
promoter
DNA sequence recognized by RNA polymerase for binding and transcription
+1 site
base pair at which initiation of transcription takes place
RNA polymerases
I: transcribes mostly rRNA
II. transcribes protein coding genes (this is what we are dealing with!)
III: transcribes mostly tRNA genes
what is the first nucleotide in pre-mRNA
nucleoside 5’ triphosphate
how is RNA pol II activated
by phosphorylation. (at end which is highly enriched in serine)
how is RNA pol II deactivated
polyadenylation signal and RNA pol II gets released from DNA template and returned to hypophophorylated stated
TATA box
consensus sequence near the promoter of many genes
TFII
Transcription Factor for RNA pol II. These bind DNA and each other to direct proper localization of RNA pol II for initiation of transcription at the +1 site.
PIC
pre-initiation complex. The complete ensemble of protein complexes at the txn initiation site. (TATA binding protein, TBP associated factors, mediator, etc) RNA polymerase eventually binds to these
what initiates translation
hyperphosphorylation of the carboxy-terminal domain of the largest RNA pol II subunit.
cis elements
consensus segments on DNA itself that function as binding sites for proteins that will work to increase rate of txn initiation.
trans elements
proteins that bind to the cis elements
different types of cis elements
promoter proximal elements: bound by constitutive (unregulated) txn factors are close to promoter
type of trans elements
bound by regulated txn factors and located far from promoter region
how do txn factors work
bind to specific DNA target sequences and recruit other proteins (co-activators) that affect formation of PIC of accessibility of DNA for txn initiation
HAT
histone acetyl transferase - co-activator acetylates histone lysine (+ charge) residues and recruits other proteins that have domains recognizing these tails
HDAC
histone deacetylase which has an inhibitory effect
histone signatures
particular modifications of histone proteins are read out by txn machinery to either positively or negatively regulate the rate of txn initiation.
co-activators
recruited by txn factors
- Histone modifying enzyme
- Chromatin remodeling complex
positioning chromatin remodeling complex
reposition nucleosomes on DNA to expose regulatory sequences
negative regulation occurs via
DNA methylation of C resides in C-phosphate-G dinucleotides - this recruits enzymes that alters local chromatin (ex: MeCP2)
MeCP2
protein that binds 5 methyl cytosine and recruits a histone deacetylase
1) MeCP2 binds methylated DNA
2) HDAC recruited
3) histone deacetylated – turned OFF
epigenetic
changes to chromatin that are NOT changing the sequence. these are heritable!
5 ways to regulate txn factors
Post TLN modification Proteolysis Localization Ligand binding Synthesis
KLF1
txn factor that turns on B globin gene expression. ALSO turns on BCL11A txn repressor which turns OFF gamma
when do you have beta globin and when do you have gamma
gamma is fetal - goes down
beta is adult - goes up
alpha is fairly constant
where is KLF1 expressed
chromosome 19
when is KLF1 gene expressed
in erythropoiesis program at progenitor cell stage. Modified post translationally by phosphorylation at T41 and is inactive without this phosphorylation.
binding site sequence for KLF1
CACC box - found -90 of beta globin promoter and multiple KLF1 binding sites are in an enhancer sequence 100 kb upstream
LCR
Locus control region - KLF1 binding sites in enhancer sequence 100 kb upstream of promoter
what role does histone acetylation play in B globin
HAT opens up chromatin structure in vicinity of B globin gene promoter, allowing txn to increase 1000 fold
HbF composition
two alpha and two gamma chains.
what is negative regulation of the gamma globin gene accomplished by
activity of BCL11A, a txn repressor. This is regulated by KLF1 which means KLF both turns ON beta globin expression and turns OFF gamma globin expression!!!
HPFH
hereditary persistance of fetal hemoglobin. Gamma chains continue to be expressed at a significant level - this limits tendency of HbS(sickle) to polymerize and pts with >15% HbF show few signs of disease! Also helpful with B-thalassemia who have poor expression of B globin
locus for individuals with HPFH
contained BCL11A gene on chromosome 2 - single nucleotide polymorphisms that were correlated with HPFH patients included a number on the 2nd intron of this gene which is an enhancer sequence - thus, changes in this enhancer sequence resulted in reduced BCL11A expression and therefore continued txn of gamma globin, even into adulthood.
cap at 5’ end of mRNA
guanosine nucleotide that has been modified to contain a methyl group at the 7 position on the purine ring. This is linked to the 5’ end of the transcript in an unusual 5’ to 5’ phosphodiester bond
what is capped
mRNA
function of cap
serves as a recognition site for binding of proteins that recruit a ribosome and protects pre-mRNA and mRNA from degradation by exonucleases that chew in the 5’ to 3’ direction.
poly-A tail
at the end of the 3’ UTR sequence, a poly A signal (AAUAAA) signals for endonucleolytic cleavage 10-30 units downstream. poly-A polymerase acts on 3’ hydroxyl that adds 100-200 A residues in a template independent fashion.
PABP
poly a binding protein. protects 3’ end from degradation by 3’ to 5’ exonucleases
what do introns start with
GU dinucleotide and end with AG dinucleotide. recognized by loose consensus sequences.
branch point
near 3’ splice site, A residue in the context of loose consensus sequence
SNRP
small nuclear ribonucleoprotein particle - part of spliceosome.
reactions in splicing
2 transesterification reactions (one phosphodiester bond replaced with another)
overview of splicing rxn
- attack of branch point A OH group on the 5’ splice site - get 2’ to 5’ phosphodiester bond
- attack of 3’ OH end of the upstream exon on the 3’ splice site which joins upstream and downstream exons in standard 3’ to 5’ phosphodiester bond.
ESE sequence
in exon - exonic splicing enhancer sequences - RNA boxes bound by SR proteins (rich in serine and arginine). Correct GU and AG of intron (even though there are lots!) can be recognized in the context of these
think “SPLICE REGULATORY!!” for SR protein
what does spliceosome recognize
actually recognizes EXONS but cleaves introns out. this is why we can have very long introns that definitely contain an AG or GU in their sequence somewhere but won’t get cleaved there!
ESS
exonic splicing silencer – attach to ESE sites and make it so that the exon isnt spliced out and included in the protein. allows for many many genes to be coded from same transcript.
nonsense codon
premature stop codon
NMD
nonsense mediated decay - rapid, target degradation of mRNAs
