Offner RNA Flashcards
Difference in RNA processing for eukaryotes vs prokaryotes
Prokaryotes have no compartments for processing, eukaryotes do
What % of RNA is tRNA, rRNA and mRNA
95% tRNA, rRNA
1-5% mRNA
The template strand is the coding or non-coding strand
Non-coding
Structure of prokaryotic RNA Pol
- beta subunit
- sigma subunit
- core enzyme: alpha2, beta, beta’
Prokaryotes
Structure of RNA Pol holoenzyme
alpha2, beta, beta’, sigma
Prokaryotes
Beta subunit of prokaryotic RNA Pol
Contains catalytic site
- synthesizes 3’-5’ phosphodiester bonds
Prokaryotes
Sigma subunit of prokaryotic RNA Pol
Recognizes promoter regions
- initiates transcription
- binds -10, -35
Prokaryotes
Does bacterial transcription require a primer?
Nahh bb
Prokaryotes
Where is the prokaryotic promoter region written, and what two sequences does it consist of?
On the sense strand
- non-transcribed strand
- coding strand
consists of -35 and -10
Prokaryotes
-35 region sequence
TTGACA
Prokaryotes
Pribnow sequence (-10)
TATAAT
Prokaryotes
What is the importance of the distance between the Pribnow and -35 region
Allows for the efficient intiation of transcription
Prokaryotes
How does prokaryotic transcription start
The core binds to random promoter and non-promoter regions
- sigma binds core, if bound to non-promoter, RNA Pol released
- holoenzyme opens DNA 12bp
Prokaryotes
2 different NT binding sites of beta subunit of RNA Pol
1st for Purine
- creates different phosphodiester bond
2nd for all subsequent dNTPs
Prokaryotes
Rho-independent termination
RNA Pol slows down in GC-rich palindrome region
- Hairpin forms, NRA released due to weak following A/U bonds
- must end with UUUUU region
Prokaryotes
Rho-dependent termination
Rho contains helices, ATPase
- Rho waits for RNA Pol to slow down
- Rho moves to 3’ end of RNA by hydrolyzing ATP
- unwinds DNA/RNA to release RNA
Prokaryotes
Actinomycin D
Initiation and elongation inhibitor
- intercalates in major groove to prevent unwinding
- effective in both prok and euk
- also doxorubicin
3 eukaryotic polymerases
1) RNA Pol I
2) RNA Pol II
3) RNA Pol III
Which RNA Pol transcribes tRNAs?
RNA Pol III
Which RNA Pol transcribes mRNAs?
RNA Pol II
Which RNA Pol transcribes miRNAs, snRNAs?
RNA Pol II
Which RNA Pol transcribes the rRNA for the 4 subunits?
RNA Pol I: 28s, 18s, 5.8s
RNA Pol III: 5s
Where are RNA Pol 1,2,3 located?
RNA Pol I in nucleolus
RNA Pol II and III in nucleus
Which RNA Pol transcribes small RNAs
RNA Pol III
RNA Pol II structure
12 subunits
- core promoter binds to 3 regions
3 regions core promoter binds to
Basal (site of transcription initiation) , Constitutive (rate), Inducible (response to environmental elements)
Where is the enhancer region located
Anywhere upstream of the core promoter
Does formation of the basal transcription complex require ATP?
Yes, requires ATP hydrolysis
Components of the basal transcription complex
TBP
TAF
TFII (A,B,F,E,H)
TBP
TATA box binding protein
TAF
TATA associated factors
TFII
Transcription factors for Pol II
Which kinase phosphorylates Pol II
TFII H
How does eukaryotic transcription elongation start?
TFIIF and Pol II detach from the basal transcription complex
What occurs during termination
Dephosphorylation of Pol II and TFII F complex
Initiation of RNA Pol I
UBF and CBF combine to causes a conformational change in the DNA which allows RNA Pol I to bind and initiate transcription
UBF = Upstream Binding Factor
CBF = Core Binding Factor
Initiation of RNA Pol III
Starts on internal promoter (within the gene)
- TFIII B binds upstream, C and A bind by the +1
- Pol III binds, TFIIII C/A release, transcription initiated
Amanita Phalloides contains alpha-amanitin. Why is it dangerous?
Present in eukaryotes only
- Fully inhibits RNA Pol II, and inhibits RNA Pol III a little
- no mRNA, no proteins, liver failure, death!
What is hnRNA
mRNA without any modifications
Where do mRNA modifications take place
Nucleus, required for exit into the cytoplasm
mRNA 5’ cap
Backwards GTP added to 5’ end
- 5’5’ tri-P linkage
- 2 from mRNA attach to one on GTP
Guanylyl Transferase
Removes gamma P from RNA and PPi from reversed GTP to add 5’ cap to mRNA
3’ polyA tail
specific endonuclease cleaves 11-30 NTs downstream of AAUAAA signal region
- PolyA polymerase hydrolyzes ATP and adds 200 A residues to mRNA
Splicing mechanism
1) U1 RNA base pairs with 5’ splice junction
2) U2 binds branch site
3) U4/5/6 bind to bring intron 1 splice site near branch point
4) U1/4 leave, U6 binds to U2
5) 2’ OH of A attacks 5’ splice site
6) 3’ OH of exon 1 attacks 3’ splice site
7) exons are joined, intron leaves
Why does splicing occur
Different sets of RNA binding proteins in different tissues
How does Thalassemia occur
A new 3’ splice site results from point mutation
- stop codon brought forward
- truncated protein
How is tRNA produced in prokaryotes
As multimers
- cleaved by RNAses at 5’ and 3’ ends
- bases get modified
- CCA added to 3’ end by tRNA nucleotidyl transferase
How is tRNA produced in eukaryotes
Monomeric
- contains intron to prevent premature mRNA binding
- removed by endonuclease and ATP-dependent ligation
How do the ribosomal subunits assemble
Self-assemble
Mechanism by which eukaryotic ribosomes are formed
Precursor is spliced to 45S, which self-splices to 28S, 18S, and 5.8S
- 28S, 5.8S and 5S combine to make large subunit (60s)
- 18S becomes 40S
- small and large subunits enter cytoplasm separately
- 5S comes from RNA Pol III
Small and large subunits only combine if they are actively transcribing
Prokaryotic ribosome combos
25 + 5 = 50
16 = 30
Total = 70S
Eukaryotic ribosome combos
28:5.8 + 5S = 60
18 = 40
Total = 80S
Structure of rRNA precursor
one of each subunit rRNA
Where do all the eukaryotic rRNA precursors combine to make the large and small subunits?
Nucleolus
