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Lecture 2

BIO230 lecture 2 notes.docx

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University of Toronto St. George
Darrel Desveaux

BIO230 lecture 2 notes - Recall that the central dogma dictates that the genome is transcribed into the transcriptome that is then translated into the proteome. The players in these steps are DNA for the genome that is transcribed by RNA polymerase into RNA which is then translated by ribosomes into proteins. Each of these steps is highly regulated. - Transcriptional regulation is crucial for a number of reasons: 1. It is crucial for organisms to respond to their extracellular environment/stimuli, which is important for multicellular AND unicellular organisms; you need to know how to respond when dealing with a stress (like drought or extreme heat) in order to survive. Cells also need to know how to response to extracellular cues like hormones appropriately. These responses usually involve transcriptional responses and alterations in the RNA levels. 2. It is also important for defining cell types. In a multicellular organism, each cell type in different tissues has different functions and is structurally different. Differences in their transcriptome is what causes these different functions to arise; they express different parts of the same genome in order to carry out their different functions. a. It also defines different cell types in terms of disease states. The microarray is of cancers from different tissues, and it represents about 2000 genes and how they are expressed in different cancer types. What you will find (green = downregulation, red = upregulation) across a horizontal line is that each cancer type has a different gene expression. These can be used as a fingerprint for identifying different types of cancers. - The main player of transcription is RNA polymerase, which transcribes DNA into RNA.  It binds to DNA, starts to unwind DNA in the active site of RNA polymerase and then starts to polymerize an RNA molecule that is complementary to one strand of DNA (template strand).  The RNA molecule is synthesized from nucleotides in the form of ribonucleoside triphosphates that are added to this RNA molecule; these are dictated by the sequence of the DNA as they are complementary to specific bases in the DNA. It is the energy in the phosphate bonds that are found in ribonucleoside triphosphates that provides the energy for the catalysis or polymerization of RNA.  As the RNA polymerase moves along the DNA, the RNA molecule comes out as it is being synthesized, and there is this region of a short DNA-RNA helix that moves along with the RNA polymerase as it is transcribing this gene that is about 9 nucleotides long. - Prokaryotic transcriptional regulation  In prokaryotes, RNA polymerase requires another protein called sigma factor in order to transcribe genes.  The sigma factor helps RNA polymerase find the right location on the DNA to start transcribing.  This is called the promoter region, which is the transcriptional start site; it is where RNA polymerase will start transcribing a gene.  The sigma factor and RNA polymerase together form an RNA polymerase holoenzyme.  Once it has found the promoter, the RNA polymerase starts to unwind the DNA and can start transcribing the gene.  This process is quite inefficient; the RNA polymerase will start transcribing, fall off, then get back to transcribing, and fall off again  this is referred to as abortive initiation. It is still unsure why this happens.  At some point, RNA polymerase will transcribe approximately 10 nucleotides and then it will shift into high gear and becomes highly processive at transcribing that gene. It will normally transcribe the entire gene until it reaches a termination sequence.  So you get transcription elongation and then transcription termination a.k.a the end of transcription of that gene, which usually occurs in prokaryotes when the RNA polymerase reaches a specific DNA sequence that indicates that it needs to stop transcribing that gene. - Genes are transcribed at different efficiencies; different genes are expressed in very different rates, resulting in very different abundances in their corresponding transcripts. Gene B (refer to slide 45) is expressed at a very low level relative to Gene A. - It turns out that the fundamental principles are the same for both prokaryotic and eukaryotic organisms: gene expression in both is regulated by gene regulatory proteins, also known as transcription factors.  They bind specifically to regulatory regions of DNA.  These are called cis-elements. These are proteins that bind to specific regions of DNA.  These cis-elements can be found in different locations relative to the genes that they regulate.  They can be upstream of the gene (like promoters) and that is often where they are in prokaryotes.  In eukaryotes, it is more complicated. The cis-elements can be found upstream, downstream, very far from the gene they are regulating, or even within the gene in introns.  Now once a gene regulatory protein binds to a regulatory region of DNA, it can have mainly 2 different effects:  It can turn the gene on. These are positive regulators called activators.  It can turn the gene off. These are negative regulators called repressors. - One of the best characterized prokaryote is E.coli. it is notorious for causing gastrointestinal diseases or infections. There are many different strains of E.coli and the one used in the lab is not dangerous/ non-pathogenic.  It is a unicellular prokaryote with one chromosome of circular DNA.  It encodes about 4300 proteins and many of these are transcriptionally regulated by food availability; this will be seen through the Trp and Lac operon examples in the lecture.  It has a doubling time of 20 minutes when there is optimal food and it is taken up in an optimal way.  A prokaryotic feature: multiple genes can be transcribed into a single RNA molecule and these genes are called an OPERON.  The genes found in an operon will be adjacent to one another on the chromosome.  They are all going to be transcribed by one promoter that is regulating the expression of all of these gene and that promoter will transcribe these genes into one RNA molecule.  This is very rare in eukaryotic organisms. Most eukaryotic genes are transcribed into its own RNA molecule; you don’t have multiple genes on an RNA molecule. - Example 1: the tryptophan operon  This is basically an on/off switch that E.coli uses to monitor for tryptophan in the environment.  This operon encodes 5 genes.  These 5 genes encode enzymes for tryptophan biosynthesis.  These 5 genes are located adjacent to one another on the chromosome of E. coli. They are all regulated by one promoter and are all going to be transcribed onto one RNA molecule.  This is very efficient!  Upstream of the genes is the promoter region where RNA polymerase will bind along with sigma factor and initiate transcription of the Trp operon.  The Trp promoter also has another sequence, which is a cis-regulatory element called the operator, which is found within the promoter. The operator is bound by what is called the Trp repressor.  To ensure that E.coli only synthesizes tryptophan when there is none in the environment i.e the biosynthetic pathway for the amino acid tryptophan will only be turned on when the bacteria doesn’t have any in the environment, the promoter region can be found in 2 protein bound states:  In the first one, the promoter is bound by RNA polymerase, and this allows for transcription of tryptophan to occur; the Trp operon will be on.  In the second one, the promoter is bound by Trp repressor protein, which binds to the operator region within the promoter. This inhibits the transcription of the Trp operon and so, expression will be off.  When Trp repressor binds to the operator, it blocks the access of the promoter to the RNA polymerase. This
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