Lecture 4. Post-transcriptional circuitry Flashcards
What can be used as targets for gene regulation?
Post-transcription transcriptional processes
What determines the steady state levels of mRNA and proteins?
Synthesis rate - Degradation rate
What organism does not have trans-acting RNA elements?
S.cerevisia (does not exist in them)
What is an example of a trans-acting RNA element?
MicroRNAs
What are examples of cis-acting RNA elements?
Aptamers – natural and synthetic
Aptazymes (structures in RNA that cleave RNA, self-cleaving)
How do microRNAs become matire mRNAs?
MicroRNAs are transported out of the nucleus into the cytoplasm and their dicer processes them into mature mRNAs
What is an example of an organism that has a functional miRNA pathway?
Unicellular alga Chlamydomonas reinhardtii
What can riboswitches be reengineered for?
The purposes of synthetic biology. This serves to illustrate the distinct features of mRNA-targeted regulation
Where are riboswitches found in the body?
Widespread
What do riboswitches allow?
Metabolite control of (primarily) translation
What molecule is normally required for translation in E. coli?
Adenine (metabolite recognition system)
In M6, what molecule can be used instead of adenine to allow translation to occur?
Ammeline (specificity of recognition had changed)
What is the purpose of ligand-binding aptamers?
They provide very useful systems for controlling translation
What aptamer is responsible for mediated translational suppression in S. cerevisiae?
Tetracycline
When does aptamer mediated suppression take place?
Regulation not at transctiption level, but at translation level via mRNA
How can a protein translational repressor be engineered?
If a tight-binding RNA-binding protein is targeted to the 5’UTR of a eukaryotic mRNA, this can block scanning of the 43S ribosomal pre-initiation complex. This is exemplified here by the bacteriophage MS2 coat protein (MCP) binding to its stem-loop binding site inserted into the 5’UTR of a reporter mRNA.
What does UTR mean?
Untranslated regions
How can an engineered protein translational repressor be made regulatable?
Via a degron
Engineering of a fusion between a degron (auxin-inducible-degron; AID) and the MCP repressor protein allows the fusion protein to be degraded in response to the addition of an auxin (IAA).
What is the function of degrons?
Degrons initiate degradation of proteins, activity can be linked to ligand-binding
How can you create a posttranscriptional NOT gate?
Degron-fusion-encoding mRNA regulated by input of ligand or light
You can switch on or off the repression functionally by introducing light or a ligand depending on the degron
Constitutive promoter drives unregulated production of mRNA that is translated to form a degron-repressor fusion protein
What is an example of a used for a posttranscriptional NOT gate when the output is a repressor protein?
Can be used as an inverter device?
What happens to the translation of a repressor-encoding mRNA when tetracycline is added?
Adding tetracycline stops the repressor protein production, switching on the downstream gene as repressor synthesis switched off (NOT gate)
What is an example of a posttranscriptional AND gate in yeast?
Bacteriophage MS2 repressor gene (MCP) on the left hand side combined with degron 1. Addition of auxin can destabilise the repression and allows expression of the translation of the target gene downstream.
PCP: another bacteriophage RNA binding protein that is independent and different from MS2 and fused to a different degron. Therefore, one could be degraded in response to auxin and one could be degraded in response to light.
What is required to produce an output from an AND gate?
A and B output
What occurs in Bimolecular Fluorescence Complementation (BiFC)?
Two halves, have to guide them back together and hold it in a stable association
Fluorescence is dependent upon the binding of the two halves, and the binding of the two halves is dependent upon having the C and N terminal parts brought together
How can you create a buffered switch?
The ligand input suppresses the Repressor output, which in turn allows translation of the Reporter mRNA. Two NOT gates in series gives a buffered positive relationship between the initial INPUT and the final OUTPUT
What occurs in a buffered switch?
Relationship between the input and output is a positive one, but it would delay the switching from i.e adding the ligand and getting a response at the other end
How do you get an output from a NOR gate?
Only way to get an output is when neither inputs are on
