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Lecture 3

BIO230 lecture 3 notes.docx

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Darrel Desveaux

BIO230 lecture 3 notes - Note: the first few slides are review from the previous lecture and so, I will not be writing those things down! I will be starting these notes from Slide 7. - Some regulatory elements are distant from the transcriptional start site and influence transcription. How?  It turns out that the Lac operon is also regulated by operator sequences far away from the operon.  There are 2auxiliary operators in the E. coli genome that can be bound by the Lac repressor. They influence transcription of the Lac operon through DNA looping (more common in eukaryotes than prokaryotes).  Recall that the Lac repressor is a tetramer: 2 subunits of the tetramer bind to one operator and the other 2 subunits bind to the other operator; this brings 2 pieces of DNA that are far apart in close proximity in 3D space. This DNA looping is what allows the Lac repressor bind to operator sequences far away from the operon transcription start site.  Why? It likely increases the affinity of the Lac repressor to the Lac promoter (since it is binding to 2 operators rather than one). It may also affect the concentration of the repressor in the environment.  This DNA looping is a mechanism by which cis-elements far away from the transcriptional start site can regulate a gene of interest. - Bacteriophage lambda  It is a virus that infects bacteria.  It is a simple organism: a DNA molecule encapsulated by a protein.  It lands on a bacterial cell and injects its DNA into the bacterial cell.  Once the DNA has been inserted into the bacterial cell, the phage can undergo one of 2 lifestyles using both positive and negative regulatory mechanisms together but the two proteins actually repress each other’s synthesis. The 2 lifestyles are:  Under favourable growth conditions, the lambda phage DNA will integrate into the host bacterial chromosome and start to divide with the bacterial DNA. This is called the prophage pathway. There is very minimal amount of viral gene expression.  However, when a host cell is damaged or the bacteria undergoes an unfavourable environment, the lambda phage can switch into a secondary lifestyle. This involves excision of the viral DNA from the host chromosome, the synthesis of viral proteins needed for the formation of new viruses, the assembly of these viruses, the rapid replication of the lambda phage DNA, packaging of the viruses, and then, unfortunately for the bacteria, cell lysis occurs and a large number of these viruses are released  LYTIC PATHWAY!  There are 2 regulatory proteins that are involved in switching from these 2 pathways. These 2 proteins are responsible for switching from the prophage and the lytic pathway. They are called the lambda repressor protein (negative regulator) and the Cro protein (positive regulator).  On the left side of slide 10, the prophage state is seen; in this state, the lambda repressor protein is active. The lambda repressor protein inhibits the expression of Cro (negative regulator of the activator’s expression) and is a positive regulator of its own expression. The virus is quite dormant in this state.  The lambda repressor protein occupies the operator sequence, which is in a promoter that controls both lambda repressor protein and Cro protein expression. The lambda repressor binds to the operator and blocks the expression of Cro and activates its own synthesis. Most of the bacterial DNA is not being transcribed.  In the lytic state (on the right side of slide 10), Cro protein gets expressed, turns off the repressor protein, and promotes the expression of its own DNA/RNA.  Cro occupies the operator (but in a different location than the lambda repressor) and blocks the synthesis of the repressor protein. It allows for its own synthesis. It doesn’t actively promote recruitment of RNA polymerase to the promoter but by blocking the synthesis of the repressor, it allows its RNA to be expressed and so, most of the bacteriophage DNA is extensively transcribed. The DNA is replicated, packaged, and eventually the bacterial cell will lyse and release these new phage particles.  What triggers the switch between prophage and lytic states?  It is induced by environmental conditions. Recall that under favourable environmental conditions, you have prophage life style but when it undergoes an unfavourable environmental conditions, it switches to the lytic lifestyle.  How does it detect this at a molecular level?  Under unfavourable environmental conditions, the DNA of the bacteria will become damaged.  The host bacteria has to respond to this DNA damage by expressing DNA repair proteins that can detect and repair the damage.  That repair mechanism inactivates the lambda repressor, which means it will no longer inhibit Cro. Therefore, Cro will be expressed and that will switch it to the lytic state. - Transcriptional Circuits  This is an example of a positive feedback loop. Under good growth conditions, the repressor protein turns off Cro but it activates itself. The activation of itself is a simple genetic circuit known as a positive feedback loop. You get expression of the repressor and the repressor protein promotes its own expression, causing an amplification effect.  There are a number of different types of transcriptional circuits: 1. Positive feedback loop  under good growth conditions, the lambda repressor protein turns off Cro but it activates itself, thereby causing an amplification effect.  It can be used to create cell memory.  A transient signal turns on the expression of a protein and it will positively regulate itself such that there is a lot of amplification of the protein. When the cell divides, it allows the cell to remember that there was this stress applied because a lot of the protein will be inherited by the daughter cells.  This is what occurs in the prophage state where there is a large number of lambda repressor proteins expressed. When the bacteria divides, the 2 daughter bacterial cells inherit a lot of lambda repressor proteins and that allows the lambda phage to stay in the prophage state and only turn on the lytic stage if there is a harsh environment. 2. Negative feedback loop  the gene product inhibits the gene expression. 3. Flip flop device  one p
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