7. Gene expression Flashcards
what is the enzyme that carries out transcription
DNA- dependent RNA polymerase
- multisubunit complexes
- couples ribonucleoside triphosphates (driven by hydrolysis of PPi)
Difference between polymerase in bacteria and eukaryotes
bacteria contain one enzyme that makes most of the RNA and eukaryotees contain 4 or 5
describe prokaryotic RNA polymerase
- holoenzyme: sigma subunit enables RNA polymerase to recognise promoter regions in DNA
holoenzyme
sigma subunit and core enzyme
name of template DNA
antisense/ non coding strand
DNA sequence that has the same sequence as RNA
sense/ coding strand
protein coding genes in eukaryotes and prokaryotes
structural genes
eukaryotes: transcribed individually
prokaryotes: tandem layout and transcribed together- operons giving rise to polycistronic mRNA
How does RNAP recognise the correct DNA strand and initiate at the beginnning of a gene?
Binds to base sequence promoters
in bacteria promoter regon is reocognised by sigma factor (adaptor)
What are promoters
40 bp sequence on 5’ side of transcription start site upstream of RNA starting nucleotide
written by sense strand so matches directionality of transcribed RNA
What is the number assigned to the first base pair
+1 (no zero base)
What is the pribnow box
a common sequence in the promoter region in prokaryotes
Why can we identify promoters?
RNAP’s tight binding protects the sequence from being broken down by endonucleas DNAse I
mutations can effect RNAP binding
what does RNAP holoenzyme binding lead to
DNA ‘melting’ (separation) in its vicinity
- transcription bubble
allows complementary RNA strand synth.
bubble travels with RNAP
RNAP is processive- explain
- does not dissociate from template
- 1900 bp, up to 180 rotations
- lots of reactions until it leaves
Describe the nature of transciption
feature of transcription in prokaryotes
rapid
RNA synth is initiated as often as sterically possible
protein synth can begin before RNA is completely synthesised
Do eukaryote RNAP contain a sigma factor
no
acessory proteins ID promoter and recruit RNAPs
Transcription termination in prokaryotes
- Contains series of 4-10 A•T bases
- A G + C-rich region with a palindromic sequence
The RNA transcript (‘intrinsic terminator’) forms a self-complementary “hairpin”
- causes RNAP to pause
- permits RNAP conformational change allowing termination
What happens if E.coli do not display the intrinsic terminator
need the Rho factor
- a helicase that runs along RNA until encountering a paused RNAP
- Pushes RNAP, causes rewinding of dsDNA & release of RNA
Eukaryotic RNAP
eukaryotic vs prokaryotic RNAP
eukaryotic have a larger mass and greater subunit complexity
RNAP II promoters are…
complex/ diverse
if they are selectively expressed they contain consensous sequences such as TATA box ( equivilent to Pribnow box)
25 bp upstreat from TSS
sequences that encourage transcription
enhancers
recognised by transcription factors which can stimulate RNAP II binding and mediate selective gene expression in eukaryotes
what can DNA bending do
cause an enhancer that is far from the promoter to interact with transcription-initiation complex
General transcription factors
required for RNAP II transcription
equivalent of σ factor
other gene specific factors can enhance
General transcription factors combine with enzyme and DNA to form
preinitiation complex
where are GTF targeted to
TATA box by TATA binding protein
what do eurkaryotes lack and what is done to overcome this
precise transcription termination sites
transcript processing takes place later
Inhibitors of transcription:
RIfamycin
inhibit prokaryotic but not eukaryotic transcription, side effects
Makes RNAPs stay in the promoter region, so proteins cannot be made until bacteria runs out of protein
Prevent elongation (not promoter binding /initial bond formation)
- Inactivated RNA polymerase remains bound
- blocks further initiation
Inhibitors of transcription: Actinomycin D
- Binds to duplex DNA (intercalates) RNAP and DNA polymerase cannot get past
- Inhibits DNA replication & transcription of eukaryotes & prokaryotes
- Interferes with polymerase passage
Inhibitors of transcription: death cap
contains α-Amanitin
•tightly binds to RNAP II
- blocks the elongation step
- binds beneath the polymerase’s bridge helix
post-transcriptional processing: prokaryotes
not processed much
post transcriptional processing in eukaryotes
of hnRNA
5’ cap
polyadenylated 3’ end
splicing
RNA exported from nucleus
techinically processed whilst theyre being synthesised
RNA capping
functions
RNAP contains C terminal domain
when phosphorylated it recruits capping enzyme complex
the 5’ end is modified to 7-methylguanosine (type O cap) joined by 5’-5’ triphosphate bridge
resist degradation
efficient initiation of translation
Polyadenylation
influences mRNA stability
Complex with Poly(A)-binding Protein (PABP) & prevents degradation.
intron consenscous sequences
Found in the “GU-AG” Introns- act as recognition for RNA-binding proteins.
how are introns removed
hydrolysis
what is needed for splicing
spliceosome –snRNAs and proteins = snRNP
why are introns paradoxical
mutations can lead to disease
why is splicing needed
can produce different proteins (different destination of cell and activity) by alternative splicing
