How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.
Endonucleases decrease protein levels by specifically deteriorating mRNA levels which subsequently decreases the protein levels. The mechanism of their action is as follows:
├── Cleaving mRNA: Recognize and cut specific RNA sequences │ ├── Triggering Decay Pathways: XRN1 (5′→3′) │ Exosome complex (3′→5′) │ ├── Translation Effects: Prevents ribosome translation │ ├── Specific Targeting: IRE1 for stress responses │ └── miRNA Association: Degrades targeted mRNAs
This reduction in mRNA levels directly leads to lower protein synthesis and overall decreased protein levels in the cell.
Both proteases and endoribonucleases are enzymes that degrade molecules, however proteases cut peptide bonds in proteins, whilst endoribonuclease split RNA molecules internally. Here are distinguishing differences between both:
| Proteases | Endoribonucleases |
|---|---|
| The substrate for Proteases is protein. They break down proteins by cleaving the peptide bonds | The substrate here is RNA. chain within RNA is cleaved |
| Proteases are essential enzymes that break down proteins into amino acids for digestion, regulate blood coagulation, and play key roles in cell division, growth, apoptosis, and migration. | Important for RNA metabolism, catalyzing the cleavage of RNA molecules into smaller components, playing roles in processes like RNA degradation, processing, and defense against infections |
| Chymotrypsin,Hyaluronidase etc. | RNase A, RNase T2 etc. |
How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?
Principle
Lipid-based transfection works on the principle of electrostatic interaction of DNA-transfection reagent complexes with the cell membrane.
Endocytosis
After contact, the complexes enter the cell via endocytosis, forming endosomes.
Escape
Normally, endosomes mature into lysosomes, which degrade DNA. Lipofectamine 3000 prevents this transformation, allowing DNA to escape the endosome.
Nuclear Transport
Escaped DNA is transported to the nucleus. Reagent P3000 plays a key role in this nuclear transport.
Expression
Once inside the nucleus, DNA is transcribed into mRNA by RNA polymerase. The mRNA then exits the nucleus and is translated into protein by ribosomes in the cytoplasm. The synthesized protein carries out its intended function within the cell
Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.
In poly-transfection, each cell in the transfected population basically runs a separate experiment, examining the circuit's behavior at various DNA copy counts and allowing users to study a large number of stoichiometries in a single pot reaction to see a schematic representation of the procedure.
Neuromorphic circuit is hardware network systems which, designed on the principles of neural functions. The network systems are inspired from biological neural networks.
Primary neuronal cells, such as cortical and hippocampal neurons, are highly sensitive and do not actively divide, traditional transfection methods face challenges in achieving efficient gene delivery. Poly-transfection overcomes these limitations by facilitating high-throughput gene expression analysis ensuring that each cell represents unique gene expression level
Genetic Toggle Switches
Provide a detailed explanation of the mechanism behind genetic toggle switches, including how bi-stability is established and maintained.
The genetic toggle switch is a synthetic biological circuit that can store one bit of data and is modeled after an electronic flip-flop circuit. Each of its two promoters regulates the expression of a repressor protein, which prevents the other promoter from working. By binding to repressors, small molecule inducers can change their conformation and lessen their capacity to bind to the promoter. In order to create two stable states, the system uses mutual repression, in which one promoter stays active while generating a repressor that inhibits the other promoter.
The bistability of genetic toggle switches is an interesting mechanism that allows these synthetic biological circuits to maintain two stable states of gene expression.
Describe at least one induction method used to switch states, including molecular signals or environmental factors involved.
A crucial regulatory protein in bacteriophage lambda, the cI repressor, also called the lambda repressor, regulates the phage's life cycle and keeps it lysogenic by inhibiting lytic genes and triggering its own synthesis.
At low temperatures (e.g., 30°C), the cI protein remains stable and actively represses its target promoter, maintaining one state. However, when the temperature is increased (e.g., 42°C), the cI repressor undergoes denaturation or degradation, losing its ability to inhibit transcription. This allows the previously repressed promoter to become active, switching the system to an alternative state
Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?
Yes, there can be limitations to these genetic toggle switches. The major limitation being integrating the toggle switches into diverse systms. The structure of these circuits may not be ideal for every cell system. The host cell has a metabolic load as a result of the production of proteins linked to toggle switches. Reduced growth rates and general cellular fitness may result from this load. more circuits in chain= more metabolic cost= more load
Natural Genetic Circuit Example
Identify and describe in detail a naturally occurring genetic circuit, emphasizing its biological function, components, and regulatory interactions.
Most common and textbook example of a naturally occurring genetic circuit is Lac operon. An operon is a cluster of genes which share a common promoter
The lac operon contains three enzyme-coding structural genes and three regulatory elements. The enzymes work together to allow E. coli to digest the disaccharide lactose, and the regulatory elements control the transcription of these enzymes The lac operon in E. coli comprises three genes: lacZ, encoding β-galactosidase, which hydrolyzes lactose into glucose and galactose; lacY, encoding permease, a membrane protein facilitating lactose uptake; and lacA, encoding transacetylase, whose role in lactose metabolism is less defined. These genes are co-transcribed, ensuring coordinated expression in response to lactose availability.
Components of Lac Operon:
Repressor (I): A coding sequence for the repressor protein. The repressor protein is a trans-regulatory element, and it’s transcription is regulated by an entirely separate set of regulatory sequences.
Promoter (P): A non-coding cis-regulatory element. RNA polymerase (RNApol) must bind to the promoter region to begin mRNA transcription.
Operator (O): A non-coding cis-regulatory element. Contains a binding site for the repressor protein I. When I is bound to the operator, RNA polymerase cannot bind to the promoter.
**Regulation
Negative Regulation (Repression):** In the absence of lactose, the LacI repressor binds to the operator, preventing RNA polymerase from transcribing the structural genes. This conserves energy by inhibiting the production of unnecessary enzymes.
Induction by Lactose: When lactose is present, it is converted into allolactose, which binds to the LacI repressor, causing a conformational change that reduces its affinity for the operator. This detachment allows RNA polymerase to transcribe the operon's genes, leading to the production of enzymes necessary for lactose metabolism.