11.RNAprocessing2

11.RNAprocessing2 - Page 1258 Figure 31-47 The sequence of...

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Figure 31-47 The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene. Page 1258
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QuickTime and a TIFF (LZW) decompressor are needed to see this picture. (1) mRNA is incubated with reverse transcriptaseto make a complementary DNA (cDNA) strand. Oligo(dTi) is used as a primer, because eukaryotic mRNA are poly adenylated. (1) Remove RNA from mRNA-cDNA hybrid by treating with alkali or an enzyme to hydrolyze the RNA, leaving the single-stranded cDNA. (1) Synthesize complementary DNA strand with DNA polymerase can The looped-around 3prime end of the first DNA strand can often be used as a primer. S1 nuclease is then used to cleave the loop. (1) Clone into vector. For efficient insertion in a cloning vector, the double-stranded DNA must have single-stranded tails that are complementary to those of the vector. Making cDNA from mRNA
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Figure 31-46 An electron micrograph and its interpretive drawing of a hybrid between the antisense strand of the chicken ovalbumin gene and its corresponding mRNA. Page 1257 Introns : I-VII Exons : 1-7 Chicken Ovalbumin Gene
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Figure 31-48 The consensus sequence at the exon–intron junctions of vertebrate pre-mRNAs. Page 1258 GU always present at 5’-end of intron AG always present at 3’-end of intron
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Catalytic Steps of Splicing Two transesterification reactions Conserved from yeast to humans OH P O O O O P OH O O O O
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Figure 31-49 The sequence of transesterification reactions that splice together the exons of eukaryotic pre-mRNAs. Page 1259 First transesterification Second transesterification
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Ribozymes RNA can be catalytic - RIBOZYMES Exhibit Michaelis-Menten kinetics e.g., K M = 42 μ M, k cat = 0.033 sec -1 RNA World Hypothesis RNA was the original biological catalyst Protein-based catalysts were relatively late additions to life
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Figure 31-51a The self-splicing group I intron from Tetrahymena thermophila . ( a ) The secondary structure of the entire 413-nt intron. Page 1261 Group I Self-Splicing Intron Secondary Structure Complete Group I Intron P4-P6 Region P: base-paired J: joining region L: loop region
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Ribozymes Catalytically active Protein independent Complex three-dimensional folds to create active site Hammerhead ribozymes : useful model system to understand ribozyme function RNAs of certain plant viruses
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Figure 31-53a X-Ray structure of a hammerhead ribozyme. ( a ) Schematic diagram with enzyme green, substrate blue, and cleavage site red. Page 1263 Hammerhead Ribozyme Secondary Structure
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X-Ray structure of a hammerhead ribozyme. ( b ) A ball-and-stick model of the ribozyme in its ground state colored as in Part a . Page 1263
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11.RNAprocessing2 - Page 1258 Figure 31-47 The sequence of...

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