Lecture 6: Termination Flashcards
What are the two types of termination?
1) Rho-independent: also known as intrinsic. No rho factors are required. Specific sequences are required for termination and these can be found with genome analysis. Found to make the end of a gene.
2) Rho-dependent: Requires rho factors. Sequence required is unknown. Distributed all over the genome.
Why is the TEC so stable?
The TEC is very stable and difficult to move once it has formed.
• Polar and VDW contacts between RNAP and the RNA-DNA hybrid backbone.
• H-bonds to ssRNA in the exit channel.
• Long range electrostatic interactions and VDWs contacts to the downstream DNA.
• Clamp closure establishes these contacts.
How does intrinsic termination work?
Intrinsic termination does not require rho factor, it instead uses a sequence which forms a hairpin as an RNA exit.
• The complex starts in the normal closed conformation.
• The inherent sequence causes pausing of the complex.
• A G-C rich hairpin loop forms.
• This is followed by a poly U tail which has weak bonds with adenine in the DNA.
• The mechanical stress breaks the weak rU-dA and causes the opening of the complex.
• The TEC complex dissociates
How does factor-dependent termination work?
Some termination sites require protein factor rho.
• Rho is a homohexameric rec-family helicase with RNA translocase activity.
• Rho binds at rut (rho utilisation) sites and then moves down the RNA using ATPase activity until it hits the paused RNAP.
• Rho is an open ring but it becomes closed when it binds to RNA.
• Rho destabilises the interactions between the template and mRNA.
• The transcript is extracted from the RNA exit channel.
What is the importance of polarity?
Polarity is a system used to measure when mRNA is not being translated (absence of ribosomes).
• It can lead to a loss of expression in an operon.
• This prevents synthesis from defective mRNA transcripts with premature mRNA transcripts.
• Normally translation and transcription occur simultaneously.
• Ribosomes will block rho from moving down to the RNA pol.
• However, if there is a premature stop codon, then the rho can move to the RNAP, as there is no ribosome.
• tRNAs can form secondary structures which prevent polarity.
• rRNAs seem to avoid rho by using antitermination.
How does NusG work?
NusG can either promote termination or elongation.
• The NTD of NusG can bind to the fork in the upstream fork junction of the TEC to promote elongation by stabilising the transcription. The CTD also interacts with an antitermination such as NusA.
• The CTD of NusG can interact with rho and promote rho-dependent termination. ChIP analysis shows that rho associates with TEC throughout transcription. It is the NusG which enhances it.
How does NusA work?
NusA can also promote either termination or elongation.
• NusA recruits NusG to the TEC.
• NusA interacts close to the RNA exit channel.
• NusA and NusG can be linked together through NusA-AR2 and NusG-AR1 as a way to link transcription and translation. This ensures elongation. NusA-AR2 can remove NusG from RNAP.
• NusA-SKK can also bind to rut sites and prevent rho binding.
How does attenuation work? Draw a diagram.
Attenuation is a method of controlling termination based upon the presence of a ligand.
• Used in the trp operon.
• When there is a lack of tryptophan, domain 1 stalls. The leader peptide cannot form. The leader peptide contains a ditryptophan (very rare).
• This leaves domains 2 and 3 free to interact and form a stem loop.
• RNA pol is free to bind.
• When there is a lot of tryptophan, domain 1 can be produced. Domain 2 also moves into the ribosome.
• The mRNA forms a stem loop with domains 3 and 4. RNA pol can’t bind, and the rest of the operon isn’t transcribed due to termination.
What are riboswitches/ribosensors?
Ribosensors/riboswitches are elements found in the 5’ UTR that control termination or translation.
• Riboswitches have a secondary structure which changes upon binding to a small molecule.
• Riboswitches can alter where the rbs is within the structure.
• Some riboswitches act as ribozymes which cleave themselves.
• They can also regulate adjacent genes which aren’t part of the same mRNA transcript.
