Question

1. Please describe negative control in terms of transcriptional regulation of gene expression?
2. Please explain the regulation of Trp operon when Trp-tRNA is available in the cell?
3. What are the classes of Transcription factors based on their regulatory responsibilities? Please explain if this distinction is absolute or not?
4. Please explain the mitochondrial protein synthesis by indicating the mitochondrial protein synthesis apparatus, the mitochondrial genetic code and so on?

5. Please explain the two hybrid assay?
5. Please explain the two hybrid assay?

Answer 1

Ans.1-

Negative control

The lac operon is under both negative and positive control. The mechanisms for these will be considered separately.

1. In negative control, the lacZYAgenes are switched off by repressor when the inducer is absent (signalling an absence of lactose). When the repressor tetramer is bound to o, lacZYAis not transcribed and hence not expressed.

Repressed lac operon

2. When inducer is present (signalling the presence of lactose), it binds the repressor protein, thereby altering its conformation, decreasing its affinity for o, the operator. The dissociation of the repressor-inducer complex allows lacZYAto be transcribed and therefore expressed.

Ans.2-

Bacteria such as Escherichia coli (a friendly inhabitant of our gut) need amino acids to survive—because, like us, they need to build proteins. One of the amino acids they need is tryptophan.

If tryptophan is available in the environment, E. coli will take it up and use it to build proteins. However, E. coli can also make their own tryptophan using enzymes that are encoded by five genes. These five genes are located next to each other in what is called the trp operon.    

The trp operon includes five genes that encode enzymes needed for tryptophan biosynthesis, along with a promoter (RNA polymerase binding site) and an operator (binding site for a repressor protein). The genes of the trp operon are transcribed as a single mRNA.  

Turning the operon "on" and "off"

What does the operator do? This stretch of DNA is recognized by a regulatory protein known as the trp repressor. When the repressor binds to the DNA of the operator, it keeps the operon from being transcribed by physically getting in the way of RNA polymerase, the transcription enzyme.

The trp repressor does not always bind to DNA. Instead, it binds and blocks transcription only when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and changes their shape so they become active. A small molecule like trytophan, which switches a repressor into its active state, is called a corepressor.

When there is little tryptophan in the cell, on the other hand, the trp repressor is inactive (because no tryptophan is available to bind to and activate it). It does not attach to the DNA or block transcription, and this allows the trp operon to be transcribed by RNA polymerase.

In this system, the trp repressor acts as both a sensor and a switch. It senses whether tryptophan is already present at high levels, and if so, it switches the operon to the "off" position, preventing unnecessary biosynthetic enzymes from being made.