Transcriptional Control - Professor Latchman Flashcards
What is transcriptional control?
This is the correct model in cells and this is where genes that are not active in a specific cell type are not transcribed.
What is post-transcriptional control?
This is where all genes are transcribed to make a primary RNA transcript but only the RNAs from the genes that are active in that cell type are processes into mRNAs which then make the proteins. The other RNAs remain in the nucleus.
Describe the evidence for transcriptional control
Doing a northern blot of the RNA in the nucleus gives evidence for a northern blot. You see precursor mRNA of the active genes, These precursor RNAs are usually longer as they have introns etc. For genes that are not active you do not see any RNAs at all in the nucleus, this shows that not all genes are transcribed into primary RNA transcripts. This is only consistent with transcriptional control and does not prove it, this is because we are only measuring the RNA produced at a set time, the RNA for the inactive genes could of been made but then degraded.
Describe Pulse Labelling
You give a quick pulse of radioactive precursors into the cell and then you ask how much of the radioactive precursor is incorporated into the RNA over a very short period of time. Over a long period of time will open the method up to the degradation criticism.
The radioactive precursor is 3H UTP, and you can measure how much of this is incorporated into the RNA, this method is the gold standard as you are measuring RNA synthesis over a very short period of time.
The results from this show that the RNA was only incorporated into certain genes for each cell type, again this is consistant with transcriptional control.
What is the issue with pulse labelling and how is this issue rectified?
You can only do pulse labelling for abundant RNAs in a cell, as only RNAs being made in vast quantities will allow enough 3H UTP to be incorporated to be labelled.
A way to get around this issue is to do a nuclear run-on assay that increases the sensitivity of the technique to allow it to include less abundant RNAs.
Describe the Nuclear run-on assay
The radioactive 3H UTPs need to get into the nucleus to interact with the RNAs but they have to pass through the cytoplasm first. The cytoplasm contains naturally occurring UTPs and this dilutes the 3H UTPs by a large factor. To prevent this you can remove the cytoplasm, this is not the gold standard anymore as you have introduced artificiality into the method. With the cytoplasm removed the 3H UTPs can go straight into the nucleus to interact with the RNA, this increases the sensitivity. When the cytoplasm is removed however, the RNA polymerases source of nucleotides is also removed. So the RNA polymerase stops transcribing, however it does not fall off. You can then add radioactive nucleotides to the isolated nucleus and the RNAs made will be radioactive. We can then take a mix of all the RNAs made by that cell type and hybridise then to the genes of the proteins we are interested in studying. The more actively transcribed a gene is the more RNA polymerases will be present on that gene.
Describe how Drosophila have provided evidence for transcriptional control
The salivary glands in drosophila larvae can amplify their DNA to create giant chromosomes. This molecule is called a polytene chromosome and consists of thousands of DNA molecule laying side by side. Under certain situations a section of the chromosome can form a puff, researchers believed that these puffs was where transcription was taking place, and the puffing was to give the transcriptional machinery space to bind. If we do a pulse labelling technique to label the RNAs transcribed and then we can hybridise back the labelled RNA to the chromosome to see where it binds and what genes were transcribed. The labelled RNAs bound to the puffed region to confirm that transcription was occurring here.
Describe an example where a steroid hormone can have two different effects in two different cell types
Oestrogen is a steroid hormone that is a short term inducer of gene expression. If you add oestrogen to a chick oviduct then the gene to make Ovalbumia is switched on. However, if you add oestrogen to the liver of a chicken then you will get the gene to make Uitellogenia switched on instead.
Describe the processing versus discard example of post-transcriptional control
In some species such as drosophila, gene expression by RNA splicing. An example of this is seen in the RNA for drosophila P element, the RNA transcript can either be spliced correctly and the RNA is translated or the cell can choice not to splice it and the RNA is degraded.
Describe a mechanism of alternative splicing in eukaryotes that leads to two different proteins in different cells
Calcitonin is a hormone that regulates the calcium levels in the body, molecular biologists isolated the gene for this protein. They found that the mRNA of this gene produced in the thyroid gland was made up of 4 exons. They new the gene had two further exons downstream of these 4 but they were not included in the mRNA. They also found that the same gene in brain cells showed different pattern of splicing. Exon 4 was missed out and replaced by exon 5 and 6, this made a protein with a different structure that acts differently, this protein was called CGRP (Calcitonin gene related protein). This one gene therefore can produce two different proteins in two different cell types due to alternative splicing. CGRP is the strongest dilator of blood vessels that we have found.
Describe how the Troponin gene shows post-transcriptional control in eukaryotes
Troponin gene is only transcribed in skeletal muscle but different types of skeletal muscle splice the gene differently to make slightly different forms of the protein Troponin with slightly different properties.
Describe how antibody molecules in eukaryotes show post-transcriptional control
The immune response needs to types of antibodies, the early type of antibodies need to be able to stick into a B cells membrane in order to trigger it to multiply that active antibody to fight infection. The late form of the antibody needs to be able to dissociate from the B cell and travel around the blood stream attacking pathogens. Alternative splicing can do this. Including an exon or not at the C terminus of the antibody effects whether it will bind to the B cell.
How can RNA stability give another level of gene control?
The amount of protein that is made in a cell often depends on how long the mRNA can stay alive in the cytoplasm. An example of this is Haemoglobin in RBCs.
Describe an example of RNA stability in gene regulation with reference to the Casein protein.
Casein is a major protein in milk and it is stimulated by the hormone prolactin. When no prolactin is present, Casein degrades very quickly and when prolactin is present it degrades very slowly. Prolactin also increases the transcription of the Casein gene. Prolactin makes the Casein RNA more stable, this is done as well as an increase in transcription because it allows a rapid response and Casein protein to be made instantly by multiple translations while the new mRNA is being made.
Describe how Histone mRNAs are made more stable
During S phase the DNA doubles and therefore Histone proteins need to double as well to wrap all the new DNA up. Histone mRNA can form a loop at its 3’ end and this stabilises it so that one mRNA can be long lived and translate many proteins. When the need for Histones is reduced the loop doesn’t form and the mRNA degrades faster.
