Weaver eoc answers 14

Weaver eoc answers 14 - Answers to Weaver end of chapter...

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Chapter 14 Messenger RNA Processing I: Splicing 1. Describe and give the results of an R-looping experiment that demonstrates that an intron is transcribed. Refer to Fig. 14.3. In R-looping experiments, RNA is hybridized to its DNA template. When the mouse β -globin mRNA precursor was hybridized to a cloned mouse β -globin gene (DNA), a smooth hybrid was formed (as opposed to when the mature mRNA was used), indicating that the introns are present in the original RNA made (the pre-mRNA). 2. Diagram the lariat mechanism of splicing. Refer to Fig. 14.4. 3. Present gel electrophoresis data that suggests that the excised intron is circular, or lariat shaped. Sharp and colleagues made a cell-free splicing extract and used it to splice and RNA with an intron. The radiolabeled transcript, derived from a portion of adenovirus major late region contained two leader exons with a (231-nt) intron in between. After incubation, the products were electrophoresed revealing the unspliced precursor plus a novel band with unusual behavior on gel electrophoresis. The novel band migrated faster than the precursor on a 4% polyacrylamine gel, but slower than the precursor on a 10% polyacrylamine gel. This behavior is characteristic of circular or branched RNAs, such as lariat shaped RNAs. 4. Present gel electrophoresis data that distinguishes between a lariat-shaped splicing intermediate (the intron-exon-2 intermediate) and a lariat-shaped product (the excised intron). Refer to Fig. 14.5. Sharp and colleagues performed splicing reactions in vitro and electrophoresed the products after various times. Bands containing the intron-exon 2 intermediate or the intron itself, could be resolved from each other and displayed anomalously low electrophorectic mobilities (being near the top of the gel and suggesting circular or lariat shaped geometries). 5. The lariat model predicts an intermediate with a branched nucleotide. Describe and show the results that confirms this prediction. Refer to Figs. 14.6 and 14.7. RNase T1/T2 and Rnase P1 cut after every nucleotide in RNA, T1 or T2 leave nucleoside-3’-phosphates, whereas P1 generates nucleoside-5’- phosphates. An ordinary mononucleotide has a charge of –2; however, RNase digestion of the splicing intermediate resulted in products (as determined by thin-layer chromatography) displaying charges of –6 and –4, from T2 and P1 treatment, respectively. These charges are consistent with a branched structure. To prove that these structures were correct, Sharp and colleagues treated the RNase P1 product with periodate and aniline to remove the 2’ and 3’- nucleosides by β -elimination. The resulting product comigrated with adenosine 2’,3’,5’- trisphosphate, verifying the presence of a branch and demonstrating that the branch occurs at an adenine nucleotide. 6. Describe and give the results of an experiment that shows that a sequence (UACUAAC)
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Weaver eoc answers 14 - Answers to Weaver end of chapter...

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