Presentation 9-9-12

Presentation 9-9-12 - Lecture 26 mRNA Stability and...

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Lecture 26 mRNA Stability and Localization Lewin’s Genes X Chapter 22 1 3 ! UTR (3 ! untranslated region) – The untranslated sequence downstream from the coding region of an mRNA. 5 ! UTR (5 ! untranslated region) – The untranslated sequence upstream from the coding region of an mRNA. stem-loop – A secondary structure that appears in RNAs consisting of a base- paired region (stem) and a terminal loop of single-stranded RNA. Both are variable in size.
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FIGURE 22.1: Features of prokaryotic (A) and eukaryotic (B and C) mRNAs.
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FIGURE 3.33 fractionation on an oligo(dT) column Poly A+ tails can be used to isolate mRNA
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Poly A+ tails are ~ 200 nucleotides in length 1) The poly A+ tail of hnRNA appeared somewhat larger than that of cytoplasmic mRNA 2) Indicates that once the mRNA reaches the cytoplasm in HeLa cells, the attached poly A+ sequences become progressively shorter over the period of time that the mRNA is used for protein synthesis. From: hnRNA nuclear From: mRNA cytoplasmic
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Effect of polyadenylation on translatability of mRNAs: VSV.N mRNAs [ vesicular stomatitis virus (VSV)N-protein ] were incubated with 35 S- methionine in a rabbit reticulocyte extracts. The mRNAs were capped (green) or uncapped (red), and poly A+ (squares) or poly A - (circles). After allowing 30 minutes for protein synthesis, the labeled products were electrophoresed and radioactivity of newly synthesized protein measured by quantitative Fuorography. D. Munroe and A. Jacobson, mRNA poly(A) tail, a 3’ enhancer of translational initiation, MCB 10:3445, 1990. Vesicular stomatitis virus (VSV) is a virus in the family Rhabdoviridae; the well-known Rabies virus belongs to the same family. VSV can infect insects and mammals.
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Effect of polyadenylation on recruitment of mRNA to polysomes. 32 P-labeled poly A+ (blue) and 3 H-labeled poly A - (red) mRNA from a rabbit reticulocyte extract, then separated polysomes from monosomes by sucrose gradient ultracentrifugation. The arrow denotes the monosome peak; fractions to the left of this peak are polysomes, and one can see the disome, trisome, and even higer polysome peaks. The poly A+ mRNA is clearly better at associating with polysomes, especially the higher polysomes. The inset shows the ratio of polyA+ to polyA - RNA in fractions 11-28. Again, this demonstrates a preferential association of polyA+ mRNA with polysomes (the lower fraction numbers). From D. Munroe and A. Jacobson , mRNA poly(A) tail, a 3’ enhancer of translational initiation, MCB 10:3445, 1990.
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Messenger RNAs Are Unstable Molecules mRNA instability is due to the action of ribonucleases . Ribonucleases differ in their substrate preference and mode of attack. endoribonuclease
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Presentation 9-9-12 - Lecture 26 mRNA Stability and...

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