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Biology 1001A
Tom Haffie

Lecture 3: Protein structure & function -Photochemistry in rods and cones of our eye -Photochemistry in a system -Photochemistry occurring in photoreceptor or in reaction centre -Why don’t we have a light harvesting antenna surrounding a photoreceptor that allows for transfer to reaction centre? Yet there is one around photosystem? -if you’re a photosystem, if your chlaymy and your photosynthetic, you simply want to harvest as much light as you can -in a photoreceptor, the arrangement of rods and cones is attempting to provide a mimic to harvest light as info; where those photons come from conveys info -photosynthetic systems don’t care about information; they care about light harvesting -arrangement of this is so you can get an image or the right environment—if very few photons, very few photons impinging on any one of these fundamental difference info vs energy of light harvesting antenna vs not having an antenna Protein structure and function -opsin is a protein -photosystem made up of 50 proteins that come together; proteins are very important -know that biological function is linked to proteins; proteins are the enzymes, and carry out most of the functional aspects of a cell -lipids= structure; carb= structure, energy source; BUT proteins are the important ones -how do you study proteins? -humans have 20 000 expressed genes and 20 000 different proteins; how do you isolate any one protein to study it? -field of biochemistry was build around the isolation of proteins from cells purifying the protein, putting it into a test tube and studying its structure which is difficult to isolate a specific protein -counter by field of genetics which was built around linking phenotypes to genotypes- discover defects in genes are linked to changes in phenotype -2 fields are very separate but are brought together by molecular biology which merged the two fields -molecular biology: isolate genes and express the gene in bacterium or eukaryotes to make the protein and study the protein; biochemistry and genetics have been brought together in that its easy to clone genes and express them today Protein abundance: points of control -what controls protein abundance? -ex. Protein hexokinase (enzyme that is required for glycolysis): why do some cells have a lot of hexokinase and others do not? -genes code for proteins; so one way to control abundance is to control transcription; copying DNA into messenger RNA is one level of control whose transcription makes hexokinase RNA; without transcription you will not get any protein -translation: mechanisms by which translation is haulted; control at level of translation of conversion of the mRNA protein; ***control from DNA to mRNA and control at level of translating that mRNA into a hexokinase protein -transcript is the product of transcription—how much specific (not total) mRNA do we have for hexokinase? What controls transcript abundance? -dependent on transcription bc transcription makes it -if transcription rates are high, transcribing lots of hexokinase gene –should make lots of transcript, but not necessarily the case bc theres another controlling step or process which is mRNA decay (important function) -when mRNA is made, doesn’t just stick around forever, some float around for 20min, or even an hour before they break down—competing process with transcription called mRNA decay/turnover -some mRNA decay quickly, some very slowly, this is controlled as is transcription -transcript abundance is the balance of these to processes—mRNA decay and transcription -how to modulate transcript abundance by changing only one process or by changing both Measuring transcript abundance -how to measure abundance of one specific mRNA within a cell? -ex. Hexokinase: -northern blot, single strand of DNA (1)isolate total RNA from cell or tissue samples; (2)run RNA on a gel electrophoresis—have tissue of human brain, heat, lung, spleen & bacterium—once you have isolated RNA its easy to quantify to see how much total RNA present make sure to load exact same amount of total micrograms of RNA in each lane so you can quantify and make sure gel has equal amounts have 20 000 mRNA and don’t see them on the gel bc their abundance is much lower; 97% of total RNA is ribosomal RNA which is projected on the bands in this example rRNA is much more abundant than mRNA—see ribosomal bands but not messenger bands; mRNA is only 3% of total RNA pool  these are the major subunits that make the ribosome and you can see the prokaryotic ribosome is different in size than the eukaryotic ribosome and is reflected by that these bands do not migrate in the same way on the gel -we have the floppy gel, transfer RNA to a nylon membrane; we can manipulate with the gel membrane -now we can probe the membrane- able to detect a specific mRNA -3% of total RNA is mRNA; of that 3% a very very small percentage is the hexokinase transcript we are looking for; but there are hundreds or thousands of copies of mRNA that codes for hexokinase (looking for something in very low abundance) -we have a probe that we label radioactively so we can detect the probe; -in the lab we have the hexokinase gene, and can make a single stranded DNA probe that would hybridize to the mRNA corresponding to that gene—that single stranded mRNA is immobilized on the membrane, we have a single strand copy of the DNA that is labeled radioactively and hybridization will take place; **the sequences are identical, hybridization takes place; the radioactive probe will stick to the membrane exactly where the complementary sequence is -single stranded DNA gene specific probe that will hybridize to a single stranded mRNA and will do so very specifically—its radioactive so we can expose this to film and end up having the abundance of that specific transcript; higher levels of hexokinase in brain then in heart -only get a signal from human tissue, not ecoli lane -don’t see any hybridization in ecoli lane?? Why is this?? Measuring protein abundance -technically easier than measuring transcript abundance -SDS-PAGE gel: aren’t pigment protein complexes so must stain the gel with blue dye; can see many thousands of proteins -we are looking for protein abundance of one protein; there are equal amounts of proteins in these 5 lanes -transfer this gel to a membrane, and probe it with a probe specific for protein we are interested in -ex probe blot for hexokinase and use an antibody for that; -antibody is usually raised in a rabbit, chicken, mouse and is specific for hexokinase and will bind to it, will detect the presence of the antibody in the lab -wash or incubate western blot with antibody to hexokinase, antibody sticks to p
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