Lecture 36: RNA Structure, Synthesis, Processing Flashcards
Transcription features
- DNA read 3’ → 5’
- RNA synth. 5’ → 3’
- No primer req.
- Coding (sense) strand = 5’ → 3’; copy of RNA
- Template (non-coding) strand = 3’ → 5’; read to RNA
Bacterial RNA polymerase structure
β subunit = catalytic site making 3’,5’ phosphodiester bonds
σ subunit = recognizes promoters to init. transcription
α subunit = structural
β’ subunit = binds DNA template
Core enzyme = α2ββ’
Holoenzyme = α2ββ’σ
RNA vs DNA
- RNA has ribose (not deoxyribose)
- RNA Uracil not Thymine
- RNA shorter than DNA
- RNA always ss
- RNA forms secondary loop + stem structures
3 steps of RNA polymerase transcription process
- Initiation
- Elongation
- Termination
Prokaryotic promoter features
- Upstream of transcription init. start site
- Written on sense strand
- Recognized by σ subunit
Transcription initiation process
- σ binds specific DNA promoters
- Transcription bubble opens (always 12 bp)
- σ dissociates as elongation starts w/ complementary bases
Specificity of RNA polymerase σ binding to promoters
- Distance between -35 and -10 sites critical; must align w/ σ
- σ DNA binding domain unmasked when bound to Core enzyme
- Core binds randomly to DNA, σ binds core (if not at promoter, σ dissociates)
RNA polymerase β DNA binding
2 nucleotide binding sites on β subunit
1. First 5’ end purine (1st P-diester bond)
2. All subsequent nucleotides
Transcription elongation process
- RNA-DNA hybrid helix ~8 bases long forms
- σ released as 12 bp transcription bubble proceeds down DNA
Transcription termination mechanisms
- Rho independent
- Rho dependent
Rho independent transcription termination
Requires GC-rich palindrome followed by UUUs
1. Palindrome folds to hairpin followed by string of UUUs
2. Polymerase slows down
3. Weak string of A-U bonds cause transcript release
Rho dependent transcription initiation process
ρ = helicase + ATPase
1. ρ binds RNA transcript
2. Polymerase slows down at GC-rich pre-termination seq.
3. ρ moves to 3’ end of transcript
4. ρ helicase unwinds RNA/DNA hybrid
5. ρ + RNA polym. dissociate from RNA
How does rifampin affect transcription?
Inhibits formation of 1st phosphodiester bond during initiation (TB treatment), binds β subunit
How does actinomycin D affect transcription?
Intercalates between DNA strands in major groove to inhibit initiation + elongation; prevents DNA strand unwinding.
Effective in prokaryotes and eukaryotes.
Would also inhib. DNA synthesis.
Eukaryotic vs prokaryotic transcription (RNA synthesis)
Eukaryotes:
1. 3 polymerases, for rRNA, tRNA, sRNA
2. No σ subunit
3. Initiation by polym.-protein interaction, not polym.-DNA
Eukaryote RNA polymerase I
Produces large rRNAs in nucleolus: 28S, 18S, 5.8S
Eukaryote RNA polymerase III
Produces small rRNAs and tRNAs in nucleus: 5S, tRNA
Eukaryote RNA polymerase II
Produces mRNA precursors: heterogenous nuclear (hnRNA) or primary transcripts, also miRNAs, snRNAs
How is eukaryotic RNA polymerase II activity regulated?
RNA polym. II interacts w/ specific proteins which bind core promoter regulatory elements to control timing/location/context of expression
RNA polymerase II Basal Transcription Complex assembly process
- TBP (TATA binding protein)
- Many TAFs (TATA assoc. factors)
- Many other TFII transcription factors
- Finally, RNA poly II
Eukaryotic promoter/enhancer regions
+1/Inr (initiator)
Promoter located on sense strand
- Basal region
- Constitutive elements
- Inducible element
Enhancer anywhere, regulates transcription but not part of promoter
RNA poly II core promoter basal element
Transcript. init. site (TATA box, -25)
RNA poly II core promoter constitutive elements
Gene specific elements (can be many), control rate of transcript.
RNA poly II core promoter inducible element
Specificity of transcript., i.e. environ. stimuli e.g. hormone response elements
Phosphorylation control of RNA poly II initiation
One TFII transcription factor in the Basal Transcription Complex is a kinase; RNA poly II phosphorylation is req. for initiation.
De-Pi stops transcription.
How do other proteins binding to promoter/enhancer regions affect transcription?
These other proteins change conformation of DNA, affecting efficiency of RNA poly II binding and transcription.
Non-protein binding factors influencing RNA poly II transcription
Chromatin remodeling, histone acetylation
How does α-amantin affect transcription?
Inhibits RNA polym. II (much more than poly III, not poly I) leading to liver failure and death
How are RNA poly I and RNA poly III regulated?
Minimally
RNA polymerase I transcription initiation process
- Upstream + Core Binding Factors bind split promoter
- RNA poly I binds
RNA polymerase III transcription initiation process
- TFIIIs bind internal promoter (downstream of +1 Inr) and upstream promoter
- Poly III binds
- Internal factors leave and transcription starts
