Sunday, June 6, 2010

Initiation of Eukaryotic transcription

We're eukaryotes, right? So let's delve into eukaryotic processes, starting with transcription.  Bacterial transcription is also pretty important, so if you would like a refresher on that topic, check out that post. Since eukaryotes are more complex, transcription will be covered in several posts in order to hammer out all the important details.
Transcription is essential for differentiating cells: all cells contain the same genome but have different expression patterns of the genome.  The differential expression of the genome creates unique protein compositions in each cell type.  While a number of functions are the same in cells and they, therefore, express many of the same proteins, cell specialization is dependent on different protein expression patterns.  For example, some proteins are abundant in specialized cells but not other types of cells.  One method for detecting which proteins are expressed in a cell is via 2D electrophoresis, which separates proteins based on their pI and molecular weight. 

The genes of the human genome are regulated temporally and quantitatively: only approximately 10,000 genes are expressed in any singular cell type.  In fact, the expression levels of nearly every active gene is different in different cell types.  Temporally, expression is regulated by many factors, including cell cycle, external stimuli, tissue types, and embryogenesis.  Genes are also regulated quantitatively, which is determined by the rate of transcription initiation and elongation, as well as the actions of activators and repressors.  Constitutive genes are those that are expressed throughout the cell cycle; inducible genes are transcribed at different levels depending on the position in the cell cycle or developmental stage.

Modulation of gene expression can occur at several stages:
  • When and how frequently a gene is transcribed
  • Splicing
  • Exporting and localization of mRNA
  • Translation initiation
  • Stability of mRNA in the cytoplasm
  •  Activation, degradation, and compartmentalization of proteins


The main method of modulating gene expression is via selective transcription.  Studying transcription can occur via analysis of cDNAs, which can be probed to determine expression levels.  Microarrays have become important for comparing the expression levels of nearly every gene in a genome.  Analysis of microarrays provides a characteristic expression pattern that can even be diagnostic.

Regulation of transcription at the level of initiation
Transcription control can be the result of different expression patterns in different cell types, developmental stages, or in response to stimuli.  As mentioned previously, changing the rate of mRNA transcription initiation is the main mechanism by which the cell regulates gene expression.  Additionally, control at the stage of transcription initiation ensures that the cell does not waste energy producing mRNAs that are not useful to it.

How do we determine transcription rate?  Via run-on transcription analysis.  To perform this type of assay, we must isolate nuclei of the cell of interest, incubate it with radio-labeled ribonucleoside (32P) and then allow the transcriptional machinery to work.  The processes are allowed to continue for a short while in order to ensure that only RNA elongation and not transcription initiation are measured.  Hybridization of the labeled RNA to specific DNA allows for quantification of its relative transcription rate, compared to standards or other genes.  

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