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Lecture 2

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 September 10 2012 Lecture 2 Dr. Lasko The first step in gene expression is transcription (transmission of information in DNA into RNA, which becomes the substrate for protein synthesis)  Prokaryotic RNA Polymerase is the enzyme responsible for creating RNA from a DNA template; it has various subunits, each with a specific function o There are 2 alpha subunits  These subunits will help build the complex, and will bind protein which regulate activity of the complex, or how likely it is that RNA pol will attach to a certain gene o There is a beta and a beta prime unit, which are very similar  These will do the actual polymerization of rNTPs into RNA o There is a sigma subunit (in this case sigma 70)  This subunit it only present during the initiation phase, and it recognized specific sequences in DNA known as promoter elements (which define the beginnings of genes)  They thus identify the beginning of genes, and therefore where transcription should start  They dissociate after transcription of the first ~10bp The DNA (in this diagram in red and blue) is sandwiched by the beta subunits of RNA Pol and RNA (green) are transcribed at this location  The alpha subunits are located away from the catalytic site of the enzyme (beta subunits) which makes sense as they do NOT play a role in actual rNTP hydrolysis  There is also an omega subunit which simply helps stabilize the complex (you can get rid of it and the enzyme will still work)  These types of structural studies are important in discovering which types of a polypeptide are responsible for a given function The sigma factor is a transcription initiation factor (in the case above sigma 70)  Sigma 70 is the most common sigma factor in E. Coli 1 th BIOL 300 September 10 2012 Lecture 2 Dr. Lasko o Housekeeping genes (genes that are always active, i.e. for metabolism, etc.) are recognized by sigma 70  Promoter elements in these kinds of genes have a sequence element which recognize sigma 70 (consensus sequence) about 35 base pairs upstream from the transcription start site o The -35 consensus site is usually a TTGACA for sigma 70 o If transcription starts at +1, sequences upstream (negative value) of that will NOT be part of the RNA  There is another consensus site, TATAAT, at about 10 base pairs upstream of the start site o These values are not exact in all genes  If this arrangement of nucleotides is present in the DNA (or something close to it), then sigma 70 will be able to bind to it o Not every promoter has this sequence, but if you compare many different genes, these sequences will be most frequent in genes that are transcribed by sigma 70  There are other sigma elements which recognize other consensus sites o E.g. sigma 54 recognizes CTGGNA (N means anything) at the -24 site (not 35) and TTCGA at the -12 site (not 10) 2 th BIOL 300 September 10 2012 Lecture 2 Dr. Lasko Here we see different genes with sigma 70 promoters; we can see that the actual promoter elements are not identical in any of them  However, we see that there is a high conservation in certain regions (-35 and - 10) and though not identical in every gene, they have high similarity Matching up DNA sequences is useful, but it won’t tell you whether or not a protein actually binds to the sequence of interest  One way to determine this is through a DNase footprinting assay: o A segment of purified DNA (in a test tube, not a cell) might have a protein bound to it, we use the footprinting to find this out o The DNA is labelled with a radioactive nucleotide at one end, and is then treated with an enzyme that cuts it at a random location  We would normal expect the DNA to be degraded to monomers, which could be run out on a gel showing only one band (the labelled monomer nucleotide) o Small amounts of DNase 1 are added at a range of concentrations, the perfect one being when each DNA molecule is cut once at a random position (this is done by using many tubes of different concentrations of DNase) o If there are no proteins which can bind your DNA sequence, you would expect a gel to show random bands of all different lengths (the DNA could have been cut at any position) o If there is a DNA binding protein present, the enzyme will NOT be able to cut the DNA where the protein is bound  E.g. in a 100 nucleotide segment 3 th BIOL 300 September 10 2012 Lecture 2 Dr. Lasko labelled at the 1 nucleotide, if there is a DNA binding protein which binds nucleotides 40-60, then there will be NO bands from 40-60 nucleotides in length on your gel (i.e. a blank spot) o In the example above, we can see the control on the left where all bands are present  The footprint in the middle is left by a protein which binds to the protein around the middle of the DNA fragment  This technique is useful for finding binding sites of specific proteins to specific fragments of DNA General principles of the regulation of transcription initiation in prokaryotes:  Regulator genes produce activator and repressor proteins o Regulation can repress translation (negative regulation) and also to activated translation (positive regulation)  Activator and repressor binding proteins bind to specific regulator binding sites (RBS) which, in prokaryotes, are usually pretty close to the promoter of the gene being regulated (10s or 100s of base pairs away) o This is NOT always the case in eukaryotes  Bacteria respond to different nutrient conditions and correspondingly adjusting their growth conditions to be able to proliferate as fast as it can (this is NOT the case of eukaryotic cells in multicellular organisms) o Transcriptional control in prokaryotes therefore are often controlled by inducers and co- repressors which re usually metabolites (e.g. Lac Operon)  These control the binding of the activators and repressors to the RBS o In the Lac operon, transcription of lactose degrading metabolizing enzymes are repressed when there is no lactose present using repressor proteins  These repressors are inhibited in the presence of lactose allowing for these genes to be transcribed In the example, this promoter is for a bunch of genes which the cell needs to synthesize tryptophan (one of the 20 essential amino acids)  This operon is controlled by a regulatory element known as the operator o An operator sits on or near the promoter  When the genes are off, the repressor is active o The repressor binds to a site between -35 and -10 and prevents the sigma 70 factor from binding to the promoter and initiation transcription o In this case, the repressor physically stops RNA Pol from binding 4 th BIOL 300 September 10 2012 Lecture 2
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