Lecture 4 Midterm Exam Notes.docx

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

BIOB11 - Lecture 4 – An Overview of Transcription, rRNA synthesis and maturation. Vocabulary Core Enzyme: the first of 2 protein subunits forming RNA polymerase  made of 5 different subunits and binds to the DNA and start transcribing in a random fashion sigma factor: one of 2 protein subunit forming RNA polymerase it directs the core enzyme to the proper site of where to begin initiation (in prokaryotic transcription) holoenzyme: Core enzyme and sigma factor combine to make a holoenzyme TATA box: At the position of -10 upstream from the start point, there is a conserved sequence that is found in almost every gene – TATAAT (see upper strand 5’ to 3’)  known as the TATA box. It is where the sigma factor binds and brings the core enzyme to start transcribing at +1 (start position) Consensus Sequence: The most common version of a conserved sequence. The TTGACA sequence of a bacterial promoter (known as the -35 element) is an example of a consensus sequence. α-amanitin: is a toxin found in some mushrooms. The three classes of RNA polymerase are classified by their susceptibility to the Alpha amanitin. Ribosomes: are large complexes that consist of several ribosomal RNA’s and many proteins Nucleolus: Clusters of ribosomal RNA genes and site of ribosome assembly Nuceolar Organizing Regions: small regions of a single chromosome that all get together at the same place inside the nucleus to form this cluster called the nucleolus. Pulse Chase Experiment: 1.Pulse: add radioactive precursor to cells for short periodGet incorporation of label into macromolecule pool. 2. Chase: wash cells to remove radioactive precursor, then examine radioactive macromolecules after an incubation period. Utility: a basic biochemical tool to “tag” a molecule or population of molecules, and determine their fate over a time course. Lecture 4 – Introduction to Transcription rRNA synthesis and maturation -Central dogma of genetics : DNA makes RNA which makes Protein - -Beedle and Tatum Experiment (1940’s) •Laid foundation of biochemical genetics •Support for ‘one gene/one enzyme’ hypothesis •B&T awarded Nobel prize in 1958 -experiment on mold -They took this mold called Neurospora and the grew it in the lab on minimal media (basic carbon source) -Neurospora can make spores, self fertilize and do everything by its self -Beadle and Tatum took the DNA of the mold and removed the genes responsible for self fertilization to see what happens -so they exposed the plant to UV rays and to help the plant, they raised it in supplemented (very rich) media. -took a reductionist approach – the plant can grow on minimal but not supplemented mediaWhy? -To find this out, they raised the plant on media of just amino acids or on media of just vitamins  the plant grew only on the vitamin and not the amino acid -This suggested that this mutant demonstrates a deficiency in vitamin biosynthesis -then (continued reductionist approach) they grew the plant on media with each individual vitamin to find the specific vitamin it needed -They found that there was a mutation in the gene involved in encoding the protein for making the vitamin panthothenic acid. Generic Biochemical Pathway -Gene 1 makes enzyme 1 which converts A to B -Gene 2 encodes enzyme 2 which converts B to C -Gene 3 encodes enzyme 3 which converts C to D -What if there was a mutation in the gene that made enzyme 2? Then if you provide the cell with C then that will rescue the mutation and bypass the block by enzyme 2. If you provide A or B it will have no effect. -So by providing the pantothenic acid, the block was bypassed and got the cells to make Co-enzyme A and grow normally. -Beadle and Tatum proposed the “One gene-one enzyme” hypothesis which states a one to one connection that a gene encodes a polypeptide of a particular biochemical function. (go to blackboard for animation on Beadle and Tatums work) Transcription -Dna makes RNA -in eukaryotic cells (it has a nucleus) -rna processed into smaller rna and some transported out of nucleus and associate with ribosomes and mRNA translates dna into protein First we will look at Transcription in Prokaryotic Cells -Fig 11-4 important know for exam!!!!! –shows how transcription elongation takes place -Transcription occurs in 5' to 3' direction BUT with respect to the strand it is being synthesized -Right side – DNA template -Left side (purple arrow) – the new RNA strand being synthesized (base pairing with complementary) in the 5’ to 3’ direction -usually only one strand is being transcribed but it starts with a double stranded template -Separating the two strands of the double stranded molecule caused topological stress which then causes overwinding of the helix which can stop the continuation of transcription. -the cell solves this problem by “supercoiling”- flipping over of the strand or coiling of the coil – to alleviate the strain -Supercoiling -the rubberband example -see figure 13-6 -The energy for the process of transcription come with the raw materials being used -NTP nucleotide triphosphate – the nucleotide bring the energy the phosphodiester bonds of the tri (three) phosphates carry a lot of energy so the cleavage of the bonds drive this process. Only one of the phosphates are incorporated in the chain; the other two are lost and recycled. -There are many RNA polymerases that can act simultaneously on the same gene at the same time. Prokaryotic Transcription -Most of what we know about transcription comes from employing Ecoli (that we have inside us) -Ecoli has one type of RNA polymerase gene and there are two part to that molecule; 1.Core enzyme made of 5 different subunits and 2. The Sigma factor -In a test tube, you put in the core enzyme and add a DNA template then the core enzyme will bind to the DNA and start transcribing in a random fashion. But this is not useful since most of the DNA is non-coding. So what the core enzyme needs is a boss or Director to tell it what to do. The director is the sigma factor to direct the proper initiation (proper site to start at). They both come together to make the complete enzyme or holoenzyme. So the sigma factor takes the enzyme to the proper site of where to begin initiation to a region close to where RNA chain will begin and it starts in a region called -10 and -35 at particular sequences conserved at those positions. -Then the sigma factor unbinds and finds another core enzyme to lead. The core enzyme begins transcription then begins. Fig 11-7 important points -double stranded DNA in antiparallel organization -Near the beginning of a bacterial gene where the start point is +1 - yellow RNA chain will begin at start point at 5’ end and continue to 3’end -The 5’ of the yellow strand is the same polarity of the top stand (also 5’ to 3’) -Area left of start point is “upstream” area right of start point is “downstream” -Upstream is where the promoter lies where the sequences that tell the gene where and when to be transcribed. These sequences attract RNA polymerase to the site so it can begin to function -Downstream is the functional gene the part that will be transcribed into RNA and then probably translated into protein -At the position of -10 upstream from the start point, there is a conserved sequence that is found in almost every gene – TATAAT (see upper strand 5’ to 3’)  known as the TATA box. AT -35 there is another conserved sequence. -Consensus sequences located at -35 and -10 in prokaryotic gene and that is where sigma factors bind -bring the core enzyme to it and the core enzyme, because of its positioning, starts transcribing at +1. -Consensus means that thing that is most often found at a particular position -Prokaryotes have one RNA polymerase but in eukaryotes, things are more complicated -There are three classes of RNA polymerase and they can be distinguished by their susceptibility to the toxin Alpha amanitin (found in some mushrooms) -RNA polymerase 1 is insensitive to amanitin because it is charged with making large RNA’s (ribosomal RNA’s) -RNA polymerase 2 is very senstive to alpha amanitin and will completelt inhibit it. RNA polymerase 2 transcribes the mRNA’s so as soon as transcription stops, the activity of the cell stops and the organism dies. -RNA polymerase 3 moderately sensitive to amanitin. Ribosomes
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