Transcription Flashcards
Prokaryotic RNA polymerase
-multi subunit enzyme which recognizes nucleotide sequence ( promoter region ) at beginning of DNA length to be transcribed and also recognizes termination sequence
What direction is RNA made
5-3 antiparallel but complimentary to template ( antisense , minus ) and identical to coding strand ( sense , plus )
How is gene to be transcribed located
-through a sequence of nucleotides near gene called promoter region
Prokaryotic RNA POL structure
-has core enzyme with 5 subunits
1 2 alpha and omega - enzyme assembly
2 Beta prime - template binding
3 Beta - 5-3 RNA polymerase activity
-has s subunit ( sigma region ) which enables RNA POL to recognize promoter region
What is holoenzyme
S region plus core enzyme
Steps in transcription ( simplified )
1 initiation
2 elongation
3 termination
What is transcription unit and transcript
- region between termination and promoter
- initial product of transcription
Initiation.
-binding of holoenzyme to template ( via beta subunit )forming closed complex and unwinding of dsDNA ( a short length approx 14 bases ) to form open complex called transcription bubble
Consensus sequence
-idealized sequence in which bases shown at each Position is base most frequently encountered at that position ie length from initiation site
( not always the case )
Sequences recognized by s factors
1 -35 consensus sequence ( 5-TTGACA-3) centered at G 35 bases from initiation site. Initial contact point for Pol and forms closed complex
Numbering of bases left and right to initiation site and reason
- negative if left and positive if right
- regulatory sequence that control transcription are designated by 5-3 sequence on coding strand
Prinbow box
- 2nd consensus site wc holoenzyme moves onto next
- (-TATAAT-) centered at middle T 10 bases away from initiation site
- melting of dsDNA occurs here to form open complex transcription bubble
Elongation
- Unwinding continues mediated by POL which then synthesizes transcript from DNA sequence
- several short RNA strands made and discarded
When does elongation begin
When transcript exceeds 10 nucleotides
What happens after transcription done at a specific gene
-s subunit released and core can leave promoter and move along template in a processive manner.
What is sliding core of prokaryotic RNA POL
Itself
What occurs during transcription between RNA and DNA
-short pieces of DNA and RNA bind forming hybrids
How are nucleotides added and you where does energy come from
- ATP dependent which is acquired from triphosphate bonds which beak releasing energy and pyrophosphate
- nucleotides added with a phosphodiester bond inbetween 3 and 5 OH via ATP hydrolysis
When does transcription stop
-at termination signal which can be spontaneous or dependent on rho proteins
Rho independent termination
-sequence in DNA generates sequence in RNA that is self-complimentary and folds in on itself with a GC rich stem and loop Making a hairpin
What is beyond hairpin
String of U’s H2 bonded to A’s on DNA strand but weak forces so facilitates separation of RNA from DNA at hybrid
Rho protein properties
Participation of hexameric Rho protein with ATPase and helicase activities
Rho dependent
-rho binds with C-rich region in rho recognition site of RNA near 5 end and uses ATPase activity to move along chain it reaches POL paused at termination site and uses ATP dependent helicase active to break hybrid zone
Action of antibiotics in prokaryotic RNA synthesis termination
- some antibiotics Prevent bacterial cell growth by inhibiting RNA synthesis
- Rifampin inhibits transcription by binding to B subunit and prevents chain extension beyond : nucleotides
- Dactinmyosin binds to DNA template and prevents movement of RNA POL along DNA
Difference between Prokaryotic and Eukaryotic Transcription
- eukaryotic cells have multiple RNA POLS for m, r and tRNA
- have transcription factors
- each RNA POL in eukaryotes has its one TF and promoters
Transcription factors structure and function
-large number of multi subunit proteins and are trans proteins
- required to bind to promoter and regulatory sequences
- bring POL 2 to promoter site
- required for transcription complex formation
Chromatin requirement for transcription
-must be relaxed to allow proteins to access DNA
Chromatin structure and gene expression
- histone modifications affect accessibility to DNA
- ATP dependent repositioning of nucleosomes is required to access histones
Acetylated and none acetylated histones
-if Acetylated then histone positive charge of leucine is cancelled and can’t bond tightly to negative DNA
Chromatin remodeling
-interconversion between euchromatin and heterochromatin
RNA POL’s of eukaryotes
1 Pol 1 - synthesis for precursor of 28S ,18S and 5.8S in rRNA in nucleolus
2 Pol 3 - synthesis of tRNA , 5S rRNA and some snRNA and snoRNA
3 Pol 2 - nuclear precursor synthesis of mRNA and also small ncRNA ( miRNA , snoRNA , snRNA )
Promoters for RNA POL 2 and function
- sequence is TATAA and similar to Prinbow Box
- -25 from transcription site
- called TATA or Hogness box
- other promoters are Inr ( initiator +1 ) or DPE ( downstream proteins +25 )
- serve as binding sites for general transcription factors ( GTF ) which interact with each other and Pol 2
Cis acting sequences
-on same gene as as sequence about to be transcribed
General transcription factors
Minimal requirement proteins for recognition of promoter , recruitment of pol 2 and initiation of transcription at basal level
-are encoded by diff genes synthesized in cytosol and transit to site of initiation so are trans acting
TFIID
GTF which recognizes and binds to TATA box and other core promoters ( DPE & Inr )
TFIIF
Brings pol 2 to promoters
TFIIH
Melts dsDNA
And clears from promoters by kinase activity phosphorylation polymerase
Regulatory elements and transcriptional factors
Are upstream of initiation site
-r additional consensus sequences
Proximal and distal regulatory consensus sequences
- close ( within 200 nucleotides ) from site of initiation
- elements such as CAAT and GC box
-further ( beyond 200 ) and include enhanced elements
Specific transcription factors
-bind to 2 domains
1 DNA binding domain
-bind to promoter proximal elements to regulate frequency of initiation
-bind to distal to mediate responses to hormones and regulate which genes are expressed
2 transcription activation domain ( recruits GTF to core promoter and coactivator Proteins
CTF and Sp1
- CAAT box transcription factor ( STF binds to CAAT box )
- binds go GC box ( specificity factor 1 )
Motifs by which TF’s bind to DNA
Zinc fingers
Leucine loops
Helix loop helix
Role of enhancers
Have sequences called response elements that bind to STF
DNA bending
-causes ehancer elements far from promoters to be closer and interact with transcription factors stimulating and regulating gene expression
Silencers
Also affect transcription from afar by inhibit it
Inhibitors of RNA polymerase 2
A-amanitin ie poison form death cap ( potent toxin ) mushroom Amaita Phalloides form tight complex with Pol 2 and inhibits mRNA synthesis s
