RNA Metabolism Flashcards
transcribed strand
template/antisense/-
nontranscribed strand
nontemplate, coding, +; identical to RNA except RNA will have U’s instead of T’s
requirements for transcription
template, all four NTPs, divalent metal ion, no primer
E. coli RNA polymerase
DNA-dependent RNA polymerase; only one type for synthesis of all RNAs
Why is RNA not proofread?
it is quickly degraded
RNA polymerase holoenzyme
core enzyme + sigma factor; must be in holoenzyme form for polymerization
sigma factor
transcription initiation factor that recognizes promoter regions in DNA and facilitates core enzyme to start transcription
promoter
where RNA polymerase binds on DNA
consensus sequence
the most common nucleotides at a particular position; practice on slide 25 if forgot
prokaryote consensus sequences
-35 (TTGACA) and -10 (TATA box)
elongation in prokaryotes
sigma factor dissociates, core enzyme proceed along DNA and EF factors bind
topoisomerase I (prokaryotes)
rewinds DNA behind the transcription bubble
topoisomerase II (prokaryotes)
releases tension ahead of the transcription bubble
quinolone antibiotics
inhibit gyrase, interfering with both DNA replication and transcription
polycistronic mRNAs
specify more than one protein; only in prokaryotes
termination in prokaryotes
Rho dependent or Rho independent
Rho independent
palindrome sequence at the end of a gene allows folding of newly transcribed RNA into a hairpin loop –> poly U stretch will pull RNA away from the DNA (not paired strongly)
Rho dependent
rho protein will bind the RNA and use its ATPase activity to separate DNA-RNA hybrid
Where does transcription occur in eukaryotes?
nucleus
mRNA
carries genetic information from DNA to ribosome, where it specifies amino acid sequence; synthesized in nucleus
rRNA
structural RNAs; synthesized in nucleolus; 80% of RNA is in cells
tRNA
transport amino acids to ribosomes for incorporation into a polypeptide undergoing synthesis; synthesized in nucleus
miRNA
small RNA molecule, which functions in transcriptional and translational regulation of gene expression
RNA polymerase I
transcribes genes in nucleolus; makes 45S for rRNAs
45S precursor becomes _
5.8, 18, and 28 (major subunit of ribosome)
RNA polymerase III
transcribes small stable RNAs, 5S rRNA, tRNAs
RNA polymerase II
transcribes mRNA precursors and miRNA
eukaryotic promoter
TATA box, located 25-35bp upstream of transcription start site
general TFs
minimal requirements for recognition of promoter; recruitment of RNA polymerase II to promoter, and initiation of transcription
sequence specific TFs
bind to proximal or distant position –> interact with core factors to modulate transcription
pre-initiation complex
TFIID binds to DNA –> kinks DNA at each end of TATA box –> other core finders bind
TFIID
TATA binding protein and TATA associated factors
TFIIH
helicase activity
steps in initiation
TFIID binds –> TFIIH has helicase activity to open DNA and phosphorylate TFIIF (RNA polymerase II)
xeroderma pigmentosa
defective TFIIH; can not perform nucleotide excision repair –> skin extremely sensitive to UV light
termination in eukaryotes
poly A complex binds to pol II and scans RNA for a polyadenylation site
noncoding genes
make up 67% of all genes
noncoding RNAs
housekeeping ncRNAs and regulatory RNAs
housekeeping RNAs
rRNA, tRNA, snRNA, snoRNA
regulatory RNAs
short ncRNAs (siRNA, miRNA) and long ncRNAs
RNA interference
dsRNA enters cell –> Dicer cleaves dsRNA into siRNA duplex –> siRNA recruited by RISC complex –> siRNA unwinds and forms protein-siRNA complex with RISC –> complex binds to target mRNA –> mRNA is cleaved at specific site and then degraded
Where does dsRNA come from?
RNA viruses, siRNA, endogenous dsRNA, miRNA, added exogenously
miRNA regulates gene expression in 3 ways
- can be incorporated into an RISC and cause degradation
- can be incorporated into an RISC and cause translational silencing
- can be incorporated into an RNA and cause silencing
RNA-induced transcriptional silencing
miRNA/siRNA trigger down regulation at specific gene –> histones modify by methylation –> heterochromatin formation
Patisiran (RNAi drug)
accumulates in livers and targets mutant transthyretin which impairs heart and nerve function
possible therapeutical uses of RNAi
antiviral therapies (knockdown host receptors), treatment for neurodegenerative diseases (reduce mutant protein levels), cancer (knockdown oncogenes)
acridine
inhibits all RNA polymerase; anti-septin
**can not be used for cancer treatment because it is too toxic to humans
alpha-amanitin
specific for polymerase II, very toxic and specific for eukaryotes
actinomycin D
inhibits all RNA polymerases; antibiotic and anti-cancer agent
rifampicin
inhibits bacterial RNA polymerase; antibiotic
antiseptic
used on the skin, contain microorganisms to deter development of bacteria and viruses
antibiotics
used inside body and only effective against bacterial infections (not viral!)
actinomycin D mechanism
binds DNA by intercalation causing DNA damage –> blocks DNA and RNA pol movement, inhibiting both transcription and replication
How can actinomycin D insert into DNA?
it has a conjugated bond and flat structure
mitochondrial transcription
monomeric RNA polymerase that has 3 promoters; creates polycistronic transcripts
mRNA processing (transcription)
5’ capping and 3’ poly A tail and splicing
5’ cap function
regulation of nuclear export, prevention of degradation by exonucleases, promotion of translation, and promotion of 5’ proximal intron excision
5’ cap reaction
phosphohydrolase cleaves gamma subunit of triphosphate –> guanylyltransferase adds a GTP –> there is now a 5’ - 5’ triphosphate linkage –> 7-methyl guanosine is added
polyadenylation
CPSF binds the RNA tail –> endonuclease cleaves 10-30 nt downstream of poly A sign, adding an -OH –> polyadenylate polymerase synthesizes poly A tail with adenyl groups coming from ATP
polyadenylation functions
increases mRNA stability, facilitate exit from nucleus, aids in translation
pre-mRNA splicing
premature RNA splices out introns to aid in formation of mature RNA
conserved splice site sequences
upstream splice site (GU), downstream splice site (AG), branch site
snRNPs
use RNA as guide to find special splicing sequence; snRNA + protein
spliceosomes
complexes of snRNPs that aid in splicing
splicing mechanism
snRNP bring the pre-mRNA, bringing sequence of neighboring exons into correct alignment –> 2’ OH group of branch site in the intron attacks the phosphate at the 5’ end of the intron –> creates a lariat structure –> 3’ OH of exon 1 will not bind 5’ at splice acceptor site of exon 2 –> releases intron lariat
lupus erythematosus
autoantibodies against the snRNPs; causes fatal inflammatory disease
ways of alternative splicing
use alternative cleavage and polyadenylation site or use different splice sites
diseases due to defective splicing
familial lipoprotein lipase deficiency, thalassemias, myotonic dystrophy type 1
familial lipoprotein lipase deficiency
LPL facilitates the removal of lipoproteins from bloodstream –> mutations causes this lipase to be inactive –> triglyceride levels build up leading to problems with pancreas and liver
familial lipoprotein lipase deficiency mutation
mutation at the acceptor site of intron 6 causes deletion of exon 7 and shifts the reading frame
familial beta-thalassemia mutation
part of intron 1 remains between exon 1 and exon 2
familial beta-thalassemia
body makes less hemoglobin, resulting in inefficient oxygenation of body
myotonic dystrophy type 1 (DM1) mutation
expansion of CTG repeats in the 3’ UTR of DMPK gene –> these transcripts are not retained in the nucleus, triggering cascade of toxic events
processing of tRNA
5’ and 3’ cleavage, addition of CCA to 3’ end
tRNA modification
methylation, deamination, reduction
mRNA editing
changes to specific nucleotides after RNA has been synthesized; rare event
example of mRNA editing
Apo B-100 to Apo B-48; Apo B-100 is in liver and Apo B-48 is edited to be put in intestine
