Exam 4: Transcription & RNA processing Flashcards
Transcription
synthesis of RNA molecules that are complementary in sequence to a DNA template
RNA is different from DNA because
it contains ribose, it uses uracil to pair with adenine, it is single stranded
RNA polymerase
unwinds template DNA & creates RNA in 5’ to 3’ direction
Initiate RNA synthesis without a primer & more prone to error than DNA polymerase
RNA polymerase I
synthesizes ribosomal RNA (rRNA), which is a component of ribosome required for protein synthesis
RNA polymerase III
synthesizes tRNA - then chemically modified to make mature tRNA
RNA polymerase II
transcribes mRNA
Promoter
start of transcription
Terminator
end point of transcription
General transcription factors
essential to allow initiation of transcription of genes by Pol II - help Pol II recognize and bind to promotors
Basal transcription complex - initiating transcription only at low rate
TFIID (transcription factor for Pol II)
general transcription factor
complex of several proteins, contains TBP - binds to TATA box
TATA box
DNA sequence area in promotor region - always found in genes transcribed by Pol II
Acts as a binding site for general transcription factor, needed for binding of Pol II
TBP (TATA binding protein)
part of TFIID; binds to TATA box
Distorts DNA and directs other components of general transcription complex & Pol II to promoter - formation of transcriptional initiation complex
High rates of transcriptional initiation require
additional factors bound to other DNA sequences - GC-rich sequences (bind transcription factor SP-1) & CAAT box (binds transcription factor NF1) & enhancer elements (located further upstream of the promoter)
Actively transcribed regions of genome are more
Relaxed than inactive regions
Death cap mushroom (Amanita phalloides)
extremely poisonous fungus
contains toxin alpha-amanitin - inhibitor of Poll II (blocks synthesis of mRNA)
Results in massive liver failure - mRNA degraded during metabolism
No antidote
Rifampicin
antibiotic, inhibitor of RNA polymerase found in bacteria
Eukaryotic Pol II is unaffected - selectively kill bacteria (antibiotic against Mycobacterium tuberculosis)
Capping
RNA processing modification at 5’ end of pre-mRNA
“Cap” structure allows cell to distinguish mRNA molecules
Important for further processing & export
Splicing
in order to form mature mRNA the introns must be removed from the pre-mRNA & exons join together
spliceosomes
catalyze pre-mRNA splicing
recognize boundaries between exons and introns
differential (alternate) splicing
a pre-mRNA molecule may contain multiple exons, different subsets of which may be spliced together generating different mRNA molecules
Gives rise to tissue-specific isoforms of enzymes and other proteins
Polyadenylation
3’ end of RNA specified by DNA signal - polyadenylation signal
transcribed into RNA & recognized by specific protein factors - cleaves RNA molecule and adds “tail” of around 200 adenine nucleotides - poly-A tail
Ribonucleases
degrade mRNA molecule from both ends - first shortening of poly-A tail, which triggers removal of 5’ cap
beta-thalassemia
results from reduced synthesis of beta-chain of hemoglobin
reduces amount of hemoglobin that can be formed = profound anemia
Phenylketonuria (PKU)
inability to convert phenylalanine to tyrosine
due to mutation in phenylalanine hydroxylase gene - single base change in 5’ splice donor site of one particular intron
Incorrectly spliced mRNA and truncated protein that lacks one exon
Chromatin remodeling complexes
use energy of ATP hydrolysis to change structure of nucleosomes so DNA becomes less tightly bound to histone core
Histone acetyltransferases (HATs)
acetylate lysine residues in histones
reduces net positive charge of histones & decreases strength of their interaction w/ DNA (negatively charged)
histones located in regions of DNA that are actively transcribed are typically hyperacetylated
histone deacetylases (HDACs)
catalyze removal of acetyl groups from histone & promote chromatin condensation
inhibit transcription
recruited to methylated DNA
DNA methylation
tends to be found in transcriptionally silent regions
Helix-turn-helix proteins
gene regulatory proteins
alpha-helices connected by short chain of amino acids
side chains of amino acids in more C-terminal of the two alpha-helices play an important role in DNA binding
homeobox proteins
Zinc finger proteins
gene regulatory protein
zinc is required for protein folding and function
alpha-helix makes contact with the major groove of DNA
glucocorticoid receptor
Leucine zipper proteins
gene regulation protein
dimers in which two alpha-helices join together to form a short coiled coil
contains hydrophobic residue, typically leucine, at every 7th position = hydrophobic residues down one side
positively-charged region interacts with DNA major groove
Fos and Jun
DNA binding proteins
act as nucleation sites - recruit more proteins
LDL receptor gene
responds to low cellular cholesterol
increased transcription of LDL receptor gene - results in increased production of LDL receptor protein and enhanced cholesterol uptake from blood
SRE-1 (sterol responsive element-1)
gene specific regulatory sequence, transcription is regulated in response to cholesterol levels
SP-1
transcription factor binds to GC boxes
zinc finger containing protein
help with assembly of Pol II
requires CRSP (cofactor required for SP-1 activation)
necessary but not sufficient for LDL receptor gene activation
SREBP-1a
leucine zipper protein that binds to SRE-1
enters nucleus when cholesterol levels fall
when bound to SRE-1 recruits HAT - relaxes chromatin & allows for LDL receptor gene transcription (as well as other genes involved in metabolism of fatty acids & cholesterol)
Binding of cortisol to glucocorticoid receptor
causes conformational change - frees receptor of associated proteins and exposes DNA-binding domain
allows uptake of hormone/receptor complex into nucleus
In nucleus binds to DNA at site called GRE (glucocorticoid responsive element) - enhancer element
GRE
enhancer element - enhances transcription
can act at a distance and found upstream, downstream, or in middle of gene it regulates
thyroid hormone receptors/RXR (retinoid X receptor) complex
found always in nucleus, bound to regulatory DNA sequences
absence of bound thryoid hormone, receptors repress transcription by recruiting HDAC activity - chromatin in condensed state
thyroid hormone binds, conformation change happens - displacement of HDAC and binding of HAT - relaxation of chromatin and transcription
Rubinstein-Taybi syndrome
intellectual disability caused by point mutations, small deletions, and rearrangements within genes encoding CBP & EP300 (which facilitate transcription of PEPCK (phosphoenolypyruvate carboxykinase) as activated by gulcagon - elevated cAMP/PKA)
CREB
PKA phosphorylates CREB, which binds to CRE promotor elements and recruits protein CBP (CREB binding protein)
CBP
CREB binding protein binds to phosphorylated CREB at CRE region of DNA - recruits EP300 = transcription of PEPCK gene
Estrogen
triggers proliferation in many breast cancers - estrogen receptor binds to DNA and recruits additional protein factors that activate transcription of estrogen-sensitive genes
Tamoxifen
competitive inhibitor of estrogen receptor
can be used to inhibit tumor growth in breast cancer
Tamoxifen/receptor complex binds to DNA still, but doesn’t recruit additional factors needed to activate transcription
