Transcription Flashcards
Elements needed for transcription?
Enzyme -> DNA-dependent RNA plymerase
Template -> DNA
Substrate -> NTPs
Phases of transcription
- Initiation: RNA polymerase binds to duplex DNA -> is unwound at promotor
- Elongation: Polymerase synthesizes RNA
- Termination: RNA polymerase and RNA are released.
Promotor
provides anchoring platform for RNA polymerase.
Contains motifs that are recognized by RNA polymerase.
4 subunits of the core enzyme of the RNA polymerase in bacteria
- (2X) α subunits (essential for enzyme’s assembly)
- β and β’ subunits (constituting the active site
responsible for RNA synthesis) - ω subunit (assembly and stability of the enzyme)
σ subunit of bacterial RNA polymerase
The core enzyme associates with the σ factor to form the RNA polymerase holoenzyme.
This makes the core enzyme specific for only binding to the promotor.
2 essential motifs of bacterial promotor
- -10 box (σ70 consensus sequence: TATAAT)
- -35 box (σ70 consensus sequence: TTGACA)
Transcription factor (TF)
Protein that controls the rate of transcription
can be activators or repressors
in order to transcribe specific genes
only when it is strictly needed
Operons
set of multiple genes under control of the same promotor
The lac operon
composed of a promoter and a operator that is recognized by a repressor protein -> lacI.
No lactose ->the repressor binds to the operator, competing with the RNA polymerase -> inhibiting transcription
Lactose present -> allolactose binds to the repressor, inducing a conformational change that impairs the ability of the repressor to bind the operator
The RNA polymerase is then free to transcribe the genes needed for the metabolism of lactose
Catabolite repression
Ensures that transcription of the lac operon cannot be turned on in the presence of glucose.
- a second messenger -> cyclic AMP (cAMP)
- positive regulator -> catabolite repressor protein (CRP)
- CRP is only active when bound to cAMP.
- cAMP is synthesized by the adenylate cyclase enzyme using ATP as the substrate
- CRP works as a dimer and is activated by a single cAMP
- Binding of cAMP causes a conformational change in CRP
- This change converts CRP from a generic DNA binder with weak affinity, into a strong DNA binder with high sequence specificity
Two main types of terminators in bacteria
- Intrinsic (or Rho-independent)
- Rho-dependent
Intrinsic terminators
- Hairpin loop is formed in the RNA
- A stretch of A residues is transcribed and forms A-U pairs
- The Hairpin is bound by NusA
- This temporarily stalls the RNA polymerase.
- The stretch of A-U pairs weakens the ineraction between the RNA and template DNA, leading to detachment of the RNA polymerase.
Rho-dependent terminators
- RNA polymerase transcribes DNA
- Rho (helicase) binds to site on RNA
- Rho moves along the RNA
- RNA polymerase pauses at the hairpin an Rho catches up
- Rho unwinds DNA-RNa hybrid
- Termination: all components are released
RNA polymerase in eukaryotes
- RNA polymerase I (rRNA genes)
- RNA polymerase II (mRNAs)
- RNA polymerase III (tRNAs)
The promotors recognized by each of these RNA polymerase enzymes have distinct strucutal features.
Transcription termination by RNA
Polymerase II
- A cleavage site is present at the end of the transcript
- The RNA Polymerase slows down on this site and an endonuclease cuts the
RNA, causing the RNA to be “released” from the elongating RNA Polymerase - A 5’-exonuclease starts to degrade the portion of RNA that is still “attached” to the RNA Polymerase (that is still elongating)
- When the 5’-exonuclease reaches the RNA Polymerase, its collision with the RNA Polymerase helps the disengagement of RNA Polymerase from DNA, leading to termination of transcription
