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Lecture 3

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 September 12 2012 Lecture 3 Dr. Lasko Some bacterial viruses subversively co-opt sigma factors and host RNA polymerase to control gene expression  Bacteria can produce their own sigma factors to re-direct the specificity of the RNA polymerase in the bacterial cell o E.g. in the bacteria (in this case bacillus), the virus (not important which one) will infect the cell, replicate itself, burst the cell and infect other cells o Some of the viral genes include proteins which will eat the cell wall to burst the cell; it also needs machinery to be able to replicate its own genome prior to the cell bursting  Viruses needs to optimize production of its own genome before it produces machinery for bursting the cell o The early genes that the virus expresses have sigma 70 promoters which “tricks” the cellular RNA polymerase into expressing viral genes  These will not immediately make the cell feel bad o One of these early genes is called gene 28 which produces a viral sigma factor which can co-opt RNA polymerase to transcribe “middle genes” needed for the viral life cycle o A middle gene called 34 encodes another sigma factor which again changes the specificity of RNA polymerase yet again to transcribe late genes which include genes meant to break open the cell wall  This temporal sequence allows for proper gene expression at a given time Transcription and translation are directly coupled in prokaryotes but not in eukaryotes  Prokaryotes don’t have a nucleus, so there are no cellular compartments and are therefore not able to separate transcription and translation  In eukaryotes, transcription happens in the nucleus and the transcripts must be are modified before they are allowed into the cytoplasm where translation occurs o There are several steps which happen between transcription and translation Prokaryotes are able to utilize mechanisms of transcriptional control which are linked to whether or not the RNA is being translated 1 th BIOL 300 September 12 2012 Lecture 3 Dr. Lasko  An example of this is Rho dependant termination (Rho being a translational terminator; we will learn about another completely different Rho later on) o Rho is a protein which binds to a specific site on the RNA termed rut (Rho utilization)  There is some sequence specificity of consensus in rut sites, but not as much as say, for example, a promoter  There are lots more potential rut sites in the genome than there are ends of genes  So how does Rho know where there is a real rut site?  Rho does not like to bind to RNA which has ribosomes on it; since transcription and translation happen at the same time, RNAs being translated may not bind Rho on this part of the Open Reading Frame (ORF)  At a real termination site, it would be located at the 3’ end downstream of the stop codon, so ribosomes will never be present there o Real rut sites are never found in the ORF, meaning that ribosomes never get in their way o Rho uses ATP to pull RNA off of the RNA polymerase separating it from the RNA DNA hybrid  Rho independent termination works through another weak consensus element which is GC rich o This GC rich region is useless in the DNA but in the RNA, this forms a stable stem loop structure  This disturbs RNA polymerase and it disturbs the geometry between the polymerase and the RNA structure, thus weakening the bond between the polymerase and the hybrid  A-T base pairs are weaker than G-C bonds, and A-U hybrids in RNA is even weaker than A-T  The sequence of T’s on the right of the consensus sequence produces a series of weak base pairs in the newly synthesized RNA strand which also helps promote transcription termination 2 th BIOL 300 September 12 2012 Lecture 3 Dr. Lasko There is also a mechanism where transcription of an operon can be terminated (or not) depending on the level of a small molecule in the cell; i.e. the tryptophan operon:  If the cell has a lot of tryptophan, the expression of the tryptophan related genes goes way down o We have seen how this works  If the cell is low on tryptophan, expression will accordingly increase  This can also be regulated through an attenuation in addition to the repressor o In high tryptophan, a 140 base pair RNA segment is made o However, under conditions of low tryptophan, a larger 7 kb mRNA including all genetic information is transcribed Within the 140bp region, known as a “trp leader region”, there is a short ORF which creates a ~14 amino acid peptide in high tryptophan levels  Notice that there are two tryptophan codons within this ORF o This in order for this protein to be synthesized, there needs to be adequate tryptophan in the cell o Thus, this mechanism can act as a sensor to detect how much tryptophan there is in the cell based on whether or not this peptide can be made  Low Tryptophan = no or little translation  High tryptophan = high transcription 3 th BIOL 300 September 12 2012 Lecture 3 Dr. Lasko  Since, in prokaryotes, transcription and translation happen in the same compartment, the secondary structure of mRNA as it comes out of transcription can affect translation in various ways o As we can see from this illustration, this 140bp RNA segment can form two different secondary structures depending on the levels of tryptophan in the cell  In high tryptophan levels, the RNA is not well transcribed and a stem-loop structure forms between 3 and 4 in the diagram  As we can see, this causes the formation of a CG rich region followed by a bunch of U’s (i.e. a transcription termination site) which stops transcription of the rest of the RNA  In low tryptophan levels, the RNA is well transcribed, favoring the formation of a stem loop between 2 and 3, which does NOT create a termination sequence and allows the rest of the gene to be transcribed Gene organization and in eukaryotes is different from prokaryotes: Genes in prokaryotes are less densely associated than they tend to be in eukaryotes; also, there is no nucleus meaning transcription and translation happen simultaneously  Extensive folding and condensation of DNA into nucleosomes and chromatin places a physical restriction on gene expression o Nucleosomes are structures formed by DNA associated with proteins called histones; the nucleosomes can then organize themselves into higher structures, finally resulting in the formation of chromatin  Genes in highly condensed regions (heterochromatin) tend not to be expressed because it is less accessible to transcription machinery  Genes in more open regions (euchromatin) are more readily expressed for the opposite reason  Much (most, in higher eukaryotes) of the DNA is non-protein-coding, both within (intronic) and between genes o In prokaryotes, ~70-80% is protein-coding, while in eukaryotes, there is a lot more space between genes, some genes encode functional RNA, some are introns and regulatory regions, etc. o We’re only beginning to understand what all of this noncoding DNA is for (it’s not just junk) 4 th BIOL 300 September 12 2012 Lecture 3 Dr. Lasko  Extensive processing of mRNA takes place in eukaryotes before it can be mature mRNA which is transcribed o 5’ cap formation o Splicing o Polyadenylation  Nuclear transport of mRNA must take place before translation can occur o Nuclear transport only happens once the mRNA has been properly processed There are various orders of DNA structure in eukaryotes:  DNA is wrapped with histones to form nucleoso
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