Transcription Flashcards
RNA polymerase action (general features of transcription)*
- begins transcription without a ‘primer’
- reads the template strand from 3’ to 5’
- synthesizes the RNA transcript 5’ to 3’ using ribonucleotides (NTPs)
- recognizes transcription termination signals
Transcription begins with
- RNA polymerase identifying a gene’s promoter region
- interacting with the DNA template strand
- initiating synthesis of a complementary, antiparallel RNA transcript
When does transcription end
When a termination signal is reached
What strand is read during transcription?
Template strand
DNA coding strand sequence compared to RNA transcript sequence
Identical except RNA has Us instead of Ts
How are are base sequences (in DNA) numbered?
By giving the first base to be transcribed a +1 designation with +2, +3, etc. in the 3’ direction (downstream to the right)
How are the upstream DNA bases to the left, in the 5’ direction designated?
-1, -2, -3, etc
Holoenzyme
Core enzyme + sigma factor
Core enzyme
4 subunits -two alpha subunits -1 beta subunit -1 beta-prime subunit =a2BB
What kind of enzyme activity does core enzyme have?
RNA polymerase activity
What is sigma factor required for?
Recognizing and binding promoter sequences
Sigma factor
- different sigma factors recognize different types of promoter sequences
- recruits the core enzyme to the DNA promoter
- dissociates from the core enzyme after the transcription begins
Initiation of transcription in prokaryotes
-scanning and recognition of promoter sequence cues by the holoenzyme trigger transcription initiation
Promoters
Contain two consensus sequences that are recognized by sigma factor
- TATA box
- -35 sequence
TATA box in prok
Consensus sequence of 6 nucleotides (TATAAT) sometimes called the pribnow box, ~7 nucleotides upstream from the +1 transcriptional start site (~-7)
-35 sequence in prok
Second consensus sequence (TTGACA) ~ 35 bases upstream (-35) from the +1 transcriptional start site
Elongation in transcription of prok
Transcription beings at the +1 transcriptional start site, sigma factor is released and the core enzyme continues
What does elongation create in transcription of prok
A temporary ‘melt’ between the double stranded DNA-forming a transcription bubble
-supercoils may be relieved by the action of topoisomerases
Net reaction of prok RNA synthesis (transcription)
Addition of a ribonucleotide to the growing RNA chain, and the release of pyrophosphate
What is pyrophospate cleaved by?
Pyrophosphatase
Cleaving of pyrophosphate by pyrophosphatase
- irreversible coupled reaction
- 2 high energy bonds are cleaved
2 termination mechanisms of prokaryotic RNA synthesis (transcription)
- Rho dependent termination
- Rho independent termination
Rho dependent termination
Requires an additional protein
-rho factor: displaces the DNA template strand from RNA polymerase
Rho factor
Displaces the DNA template strand from RNA polymerase
Rho independent termination
Requires G-C rich step loop followed by a poly-U stretch
-causes RNA polymerase to dissociate
Prokaryotic mRNA can be…
Polycistronic
Polycistronic
One mRNA can code for several proteins
Polycistronic mRNAs
Often code for multiple enzymes in the same biosynthetic pathway
Eukaryotic mRNAs are always
Monocistronic
Rifamipin (rifamyin)*
- antibiotic
- binds to prokaryotic RNA polymerase and prevents transcription initiation/elongation
- used to treat tuberculosis
Prokaryotic synthesis of tRNA and rRNA
Similar to the model for eukaryotes, except in prokaryotes the same RNA polymerase produces all types of RNA
Why is eukaryotic DNA highly condensed (chromatin)
To allow gene transcription the genes must be exposed
Euchromatin
- Less condensed
- more accessible to RNA polymerases
Acetlyation of histone H1
Causes the chromatin to revert to nucleosomes (bead on a string)
Heterochromatin
- appears more dense
- DNA is relatively inaccessible due to highly condensed structure
DNA in herterchomatin
Highly methylated
What is the foundation of epigenetics
DNA methylation
-twins with same genes, some mutations can result in the way its methylated in expressed between the twins
RNA polymerase I (euk)
Transcribes precursors to rRNA in the nucleolus
RNA polymerase II (euk)
Transcribes the precursor to mRNA (hnMRNA) in the nucleoplasm (some snRNAs as well)
RNA polymerase III (euk)
Transcribes tRNA precursors (also small rRNA and some snRNAs)
RNA pol II recognizes what two consensus sequences in eukaryotes
- TATA box (Hogness box)
- CAAT box
TATA box (Hogness box)
- similar yo prokaryotic TATA box
- approximately 25 nucleotides upstream (-25)
CAAT box
- eukaryotes
- appproximately 70 nucleotides upstream (-70)
Eukaryotic transcription elongation
Essentially the same as prokaryotic RNA syntheiss
Termination of euk transcription
Requires a signal not well understood in euk
rRNA synthesis and processing
- not actually expressing a gene
- three rRNAs are transcribed as a single larger precursor (by RNA pol I in the nucleolus)
- individual rRNAs cleaved apart by RNases
- prokaryotic rRNA synthesis is the same as in eukaryotes
- instrumental in making ribosomes
tRNA synthesis and processing
-RNA pol III in in the nucleoplasm
RNA polymerase III in tRNA synthesis and processing
- removal of intron loops
- trimming of the 5’ and 3’ ends
- base modifications
- addition of the 3’-CCA sequence***
Eukaryotic mRNA synthesis
Spatially and temporally separated from translation
Transcription takes place (euk)
In the nucleus
Translation takes place (euk)
In the cytoplasm
Eukaryotic mRNA processing
Processed extensively (hnRNA to mRNA)
When do prokaryotic mRNAs being translation?
Before transcription finishes
Shine Delgarno sequence
Tells ribosome to start translation in prok
Euk promoter regions
CAAT box (-70) TATA box (Hogness box) (-25)
Prok promoter region
TATA box (Pridnow) (-10) -35 sequence
Untranslated regions in prokaryotes and eukaryotes (UTRs)
Both have a 5’ and 3’ UTR
Coding region in eukaryotes and prokaryotes
Eukaryotes have introns and exons (monocistronic)
Prokaryotes don’t have that
5’ capping in eukaryotic mRNA
- Addition of a 7-methylguanosine to the 5’ end of the mRNA-5’ to 5’ linkage
- serves as a ribosome recognition signal and stabilizes mRNA (protects from nuclease degradation)***
3’ poly A tail in Euk mRNA
The 3’ terminus contains a polyadenylation signal- a special enzyme (poly A polymerase) adds a variable stretch of adenin residues (~40-200)
What kind of signal does the 3’ poly A tail of eukaryotic mRNA serve
Serves a signal for transport out of the nucleus, and stabilizes the mRNA from nuclease attack
Is the 3’ poly A tail terminu encoded in the gene (of euk mRNA)
No, but the SIGNAL sequence is encoded, but the additional A’s are not
Intervening sequences in euk RNA synthesis
Introns
Expressed sequences in euk RNA synthesis
Exons
Removal of introns
Must be removes from the coding region for proper translation
Splicing
The removal of introns and the jointing of exons by snRNPs
SnRNPs
Are composed of snRNAs and a group of proteins
What are the snRNPs combined with?
Primary transcript from the spliceosome
-this is the molecular machine that performs the splicing reaction
Lariat
Excised intron
Splice sites
Have very specific sequences (mutations at these sites can cause incorrect splicing)
Exons join together
During splicing to form mature mRNA
How many intron sequences can eukaryotes contain?
Between 0 and 50
Alternative splicing of hnRNAs
Joining of different exons together o form different mRNAs
What does alternative splicing permit?
The production of proteins with some common domains, but the over all function of the protein is different
-gene is same though
Systemic lupus erythematosus
Causes butterfly rash
- observed in late-teen females
- patient produces antibodies that recognize host proteins, including snRNPs
After all processing events, mature eukaryotic mRNA is transported where?
Cytoplasm
What mushroom genus accounts for 95% of all mushroom fatalities?
Genus amantia
Amanita phalloides
The death cap mushroom
Alpha-amanitin
From amanita phalloides mushroom
-binds to RNA polymerase II-inhibits mRNA synthesis
