ch 8 RNA transcription, processing, and decay Flashcards
pulse-chase experiment
method of labelling molecules with specific markers to determine where they are found
1. pulse label a eukaryotic cell with 32P UTP
2. then chase away the label with an excess of non-radioactive UTP
conclusion of pulse-chase experiment
label first appears in nucleus
after chase moves to Cytoplasm
signal is then lost (RNA turnover)
informational RNA
RNA which is used as a template for protein synthesis (mRNA)
carries the ‘genetic message’ from genes in the nucleus to the ribosome in the cytoplasm
functional RNA
RNA that is functional as an RNA molecule and is not translated into a protein
- tRNA, rRNA, snRNA, miRNA, siRNA
transfer RNA (tRNA)
transport of aa to the ribosome
ribosomal RNA (rRNA)
component of the ribosome
small nuclear RNA (snRNA)
RNA processing
microRNA (miRNA)
inhibit gene expression
small interfering RNA (siRNA)
genome integrity
gene (molecular terminology)
a segment of DNA that can be transcribed into RNA and the regulatory sequence that makes transcription possible
transcription
production of any RNA from a DNA template
typical prokaryotic gene
transcribed = coding sequence -> RNA -> protein
regulatory regions = promoter and terminator
typical eukaryotic gene
transcribed region and regulatory region
transcribed region = introns (noncoding sequence) and exons (coding sequence)
RNA is complementary to the
template strand
template strand =
non-coding strand
non-template strand =
coding strand
3 stages of transcription
- initiation - binding to template
- elongation - synthesis of RNA
- termination - release of RNA transcript
RNA polymerase
the enzyme which transcribes DNA into RNA
how does RNA polymerase know where to start transcription?
consensus sequences
- strong promoters, those with high levels of RNA transcription, are closer matches to the consensus
holoenzyme
RNA polymerase binding to promoter
sigma factor
attached to the holoenzyme
- recognizes and binds to the -35 and -10 regions
- opens helix at the -10 region (AT rich)
core enzyme
initiation
- no sigma factor
first nucleotide transcribed at +1
elongation
transcription bubble moves with polymerase
addition of new bases is always to the 3’ end of the growing chain
hairpin loop
internal base pairing of G and C forms helical stem
Poly-U stretch
follows the hairpin
- forms spontaneously
termination - intrinsic
intrinsic - factor-independent
stop point defined by newly synthesized RNA sequence of hairpin loop and ploy-u stretch
hairpin loop destabilizes DNA-RNA hybrid - RNA dissociates from DNA template
termination - rho dependent
- rho protein binds to RNA at rut site, moves towards RNA polymerase
- nearby sequence causes polymerase to pause
- rho catches up and causes RNA dissociation
why does mRNA decay
for control - respond to changing environment, pathway feedback
how does mRNA decay
5’PPP -> 5’P
endonuclease activity to cleave mRNA
exonuclease activity to degrade mRNA, processively 3’ -> 5’
activator
a protein that binds to a DNA element and activates transcription
repressor
a protein that binds to a DNA element and prevent transcription
operon
multigeneic segment of DNA sharing regulatory regions
- predominantly found in prokaryotes
transcription of eukaryotes - increased complexity:
larger genomes - comprised of chromatin
multicellular organisms
gene composition
- more genes
- significantly longer
- have non-coding regions (introns)
3 eukaryotic RNA polymerases
RNA pol I - rRNA
RNA pol II - mRNA
RNA pol III - tRNA and 5S rRNA
general transcription factors (GTFs)
needed bc RNA polymerase complex does not recognize and bind to promoters on its own
- recognize promoter elements
- may modify polymerase activity
TATA box
initiation site of eukaryotes
similar to -10 region in prokaryotes
TFIID
multi-protein complex
including TATA box binding protein
pre-initiation complex
TFIIs recruited
DNA helix opened (TFIIH)
RNA polymerase bound
what happens to get from initiation to elongation
TFIIH phosphorylates RNA pol II
RNA synthesis begins
transcriptions factors dissociate
co-transcriptional modification
mRNA processing - within the eukaryotic nucleus
5’ cap = addition pf 7-methylguanosine
removal of introns, splicing together of exons
polyA tail added to 3’ end
5’ cap
guanylyltransferase adds 7-methylguanosine to the 5’ end
poly(A) tail
endonuclease recognizes the polyadenylation site (AAUAA)
mRNA is cleaved on the 3’ side of site
50-250 A’s are added by poly
(A) polymerase (PAP)
splicing
introns, transcribed in the primary transcript, are removed and exons are spliced together
branch point
conserved sequences
- key nucleotide = A
spliceosome
an RNA/protein complex responsible for splicing
- composed of snRNA and protein = snRNPs
roles of the spliceosome
- recognize the conserved intron sequence
- help fold the intron into the correct 3d shape
- regulate the spicing process (alternative splicing)
3 steps of the 2 transesterification reactions
- 2’ OH pf branch point A attacks 5’ splice site, releases 5’ exon (3’ OH exposed)
first transesterification - A is now involved in 3’-5’ bond and 2’-5’ bond
second transesterification - 3’ OH of 5’ exon attacks 3’ splice site, joining together the 2 exons
2 methods of mRNA decay in eukaryotes
both start with deadenylation (poly(A) tail removal)
1. decapping by Dcp1/Dcp2
- 5’ -> 3’ exonuclease
2. 3’ -> 5’ exonuclease
- DcpS releases 5’m2Gp
ribozymes
RNA with enzymatic activity
- self splicing introns - free GTP attacks phosphodiester bond at 5’ splice site, 5’ exon joins 3’ exon
