Transcription factors in eukaryotes

(See figure in previous post)
Without transcription factors, RNA polymerase will not properly transcribe in vivo. In the book, there are two theories about how TF's work.

1) Transcription factors attach to the TATA box (a promoter sequence). There are many different kinds of TF's (see table 3.1). RNA polymerase then attaches to the same place.

2) TF's and RNA polymerase are already assembled, and the complex attaches to the promoter.

In both cases, the RNA is closed until a ATP-dependent process occurs and make it open. The TF's dissociate after transcription starts.

To note:
CTD: Carboxyl-terminal domain. It is a region in one end of the RNA polymerase which interacts with a protein complex called SRB/MED. SRB/MED aids in transcription

Transcription factors in eukaryotes

Transcription in eukaryotes is far more complex than in prokaryotes. This is because eukaryotes have to deal with more diverse environmental conditions. Also, multicellular organisms obviously have differences in gene expression to have its cells be of different types.

Similarities between eukaryotic and prokaryotic transcription complexes:
1) RNA polymerase is directed by a factor to the appropriate site. (Sigma factor in prokaryotes direct to TATA box). Instead of a simple sigma factor, eukaryotes use different proteins, or transcription factors.

2) RNA polymerase has to turn from closed to open state in order to transcribe

Differences:
1) Much more complex process in eukaryotes. Many more proteins acting as transcription factors. This is necessary for specificity in expressions.

2) Transcription cannot freely occur because of chromatin structure. Tightly packaged chromatin must be opened up to let transcription.

The diagram attached shows transcription factors. (In this future it will be explained, hopefully.)

DNA footprinting to check binding regions in DNA

It's important to know in what regions of DNA proteins attach. DNA footprinting can show where binding regions are located. In the experiment, DNAse is used. DNAse cuts DNA in arbitrary positions. A sequence that has no proteins binding to it will have random cuts (all different sizes in equal amounts. However, if a protein is bind to it, the protein will protect close sequences from being cut. The DNAse will only cut parts far away from the binding site. Thus, a "gap" is produced. The samples are run in gel electrophoresis.

Proteins bind to DNA, and using DNA footprinting, it can be seen where. This is possible because the protein shields the sequences near it from the DNAse.

Heterokaryons

Gene expression is partly driven by protein content of cell. Heterokaryons are fused cells. For example, a non-differentiated human cell can be fused with a differentiated mouse cell. The proteins can be seen to affect the expression on the nuclei.

RNA Polymerase in prokaryotes

Transcription in prokaryotes is simpler but very similar to that in eukaryotes. Transcription requires RNA polymerase. RNA polymerase structure contains two alpha and beta subunits. Sigma factor is needed to direct the polymerase to the TATA box. RNA polymerase in its closed form is inactive. Transcription factors attaches to the complex and opens the polymerase to an active form.

Regulatory regions in RNA II: regulations by UTR in the mRNA

UTR's are untranslated regions of a mRNA. It can be downstream or upstream of the actual mRNA. UTR's are shown to be able to regulate where genes are expressed.

Experiment from: Evans, T, Translational control of maternal glp-1 mRNA...

Expression of glp-1 mRNA is monitored in a four-celled stage of C. Elegans. Glp-1 mRNA is in all 4 cells, but only expressed in 2. A LacZ gene is put in place of glp-1, but the original UTR is left where it is. It shows then that the lacZ gene is expressed in the 2 cells where the glp-1 gene was expressed. This implies that the UTR:glp-1 regulates the location-specific translation.

Then, the UTR in that engineered lacZ:UTR-glp-1 segment is altered. As a result, the specific two cell expression of lacZ is obstructed. As the UTR is changed in structure, it shows that the UTR of glp-1 is responsible for regulation.

Conclusion: certain regions in mRNA can regulate expression, and the instruction can be found in the untranslated region (UTR) of the mRNA. Obstructing the UTR shows differences in expression, proving that the UTR plays a role in expression.

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Regulatory regions in RNA: Splicing instruction in a mRNA

A mRNA may produce multiple proteins. Alternative splicing example is where splicing instruction is in the RNA itself (so the instruction isn't limited to DNA).
Example: doublesex (dsx) gene in Drosophila, which determines sex. dsx gene has 6 exons. A specific combination produces one sex. Females are made of exons 1, 2, 3, 4, while males 1,2,3,5,6.
This shows how a primary transcript can produce entirely different proteins through different ways of splicing mRNA, making unique combinations of exons, thus making unique proteins.
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Irreversible modifications in DNA sequence: Antibody production

Antibody molecule, immunoglobin protein, is produced by a multi-step arrangement of the mRNA. For example: There are multiple V regions encoding for the variable region in a light chain. Each is labeled v1, v2, v3, etc.One of the V exons is attached to the J exon to make the final mRNA. The other V regions are lost, thus irreversible. The choosing of which v region is random. Final mRNA is perfected through further splicing.

Intro

Concepts mainly based on Molecular Principles of Animal Development by Alfonso Arias and Alison Stewart. I'm a student interested in genomics and particularly developmental biology. Reading this book is a start. By writing about the concepts and diagrams in the book, it will help me in remembering the theories.