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Lecture 5

Molecular Cell Biology Lecture 5.docx

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

Molecular Cell Biology Lecture 5 1 Eukaryotic mRNAs are synthesized as larger precursors known as hnRNAs 2 Pulse chase experiments are useful for determining how this occurs involves labeling macromolecules with radioactive NTPs separating RNAs by size by centrifugation and fractionation absorbanceradioactivity measurements follow3 Polymerase II is a large multisubunit complex which binds upstream of the gene to be transcribed Changes in the complex addition or loss of subunits phosphorylation of subunits alter its activity such that initiation elongation and termination occur properly4 A mature mRNA usually consists of a 5 cap the 5 untranslated leader the coding region the 3 untranslated region and a poly A tail5 Removal of introns occurs by RNA splicing reactions Splicing can be autonomous in the case of selfsplicing RNAs or mediated by a multisubunit complex known as the spliceosome6 Consensus sequences at the intronexon junctions are used to identify the proper splice sites7 Hybridization assays and DNA sequencing are useful for determining exactly what has occurred in mRNA maturation 332o When a eukaryotic cells are incubated for a short period in Huridine or Pphosphate and immediately killed most of the radioactivity is incorporated into a large group of RNA molecules that have the following propertiesa They have large molecular weights up to about 80S or 50000 nucleotidesb As a group they are represented by RNAs of diverse heterogeneous nucleotide sequencec They are found only in the nucleus Because of these properties these RNAs are referred to as heterogeneous nuclear RNAs hnRNAs and the red radioactivity line in Figure 1117a indicates them 332 When cells that have been incubated in Huridine or Pphosphate for a brief pulse are placed into unlabeled medium chased for several hours be fore they are killed and the RNA extracted the amount of radioactivity in the large nuclear RNAs drops sharply and appears instead in much smaller mRNAs found in the cytoplasm red line of Figure 1117b 32I Label cells for short period of time with P II Purify RNA centrifuge to separate molecules by size III Determine OD RNA and radioactivity labeled hnRNA RNALarge molecules come out first followed by the smaller moleculesThe blue lines indicate the optical density absorbance of UV light of each fraction provide information about the amount of RNA in each fraction following centrifugtationevident that most RNA in cell is present as 18S and 28S rRNAThe red lines indicate radioactivity in each fraction provide information about the number of radioactive nucleotides incorporated into differentsized RNAs during the brief pulse Evident that neither hnRNA nor the mRNA consititute significant fraction of the RNA reason is that these RNAs are degraded after relatively brief period of time Majority of RNA synthesized is hRNA but about 90 of the total RNA of a cell is rRNAo Something to keep in mind for figures 1113 and 1117 Most of newly synthesized RNA in the cell is hnRNABUT note thatMost of the stable RNA of the cell is rRNA over 90Halflifehow long before degradation or destroyed on purpose Halflife of hnRNAmRNAminutes to hours average 40min
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