ch11e-mail (2)

ch11e-mail (2) - CHAPTER 11 GENE EXPRESSION: FROM...

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C HAPTER  11 G ENE  E XPRESSION : F ROM  T RANSCRIPTION  T O  T RANSLATION Transcription: The Basic Process I. Transcription done by DNA-dependent RNA polymerases in both prokaryotes & eukaryotes; incorporate nucleotides to make RNA from DNA template ; DNA provides information for RNA strand synthesis A. The enzymes are commonly known as RNA polymerases B. They incorporate the nucleotides into the growing RNA chain one at a time; its sequence is complementary to one of the strands of DNA II. RNA synthesis begins with the association of the RNA polymerase with the DNA template – proteins have A. RNA polymerases bind to sites on DNA called promoters before starting transcription; they cannot recognize promoters on their own, but require the help of other proteins ( transcription factors) 1. Transcription factors (TFs) are particularly important in initiation of eukaryotic gene transcription B. Promoter also contains information specifying which strand of DNA to transcribe & the site at which transcription begins III. Polymerase moves along DNA in 3' —> 5' direction (toward 5' end) laying down complementary RNA in 5' —> 3' direction, unwinding DNA temporarily as it travels along DNA template A. Catalyzes reaction: RNA n + NPPP —> RNA n+1 + PP i (—> 2 P i ) 1. The ribonucleoside triphosphate precursors (NPPPs) are hydrolyzed into nucleoside monophosphates as they are polymerized into a covalent RNA chain B. Reactions leading to nucleic acid & protein synthesis are inherently different from those of intermediary metabolism 1. Such reactions must be essentially irreversible unlike many reactions of intermediary metabolism, which may be close enough to equilibrium that a considerable reverse reaction can be measured 2. To prevent reverse reaction (there is virtually none), nucleic acid synthesis is coupled to exergonic pyrophosphate hydrolysis; done by pyrophosphatase; this releases a large amount of free energy 3. Hydrolysis of pyrophosphate produces two inorganic phosphates (P I ), making overall reaction essentially irreversible 4. A nucleotide is incorporated into a RNA strand if it forms proper Watson-Crick base pair with its opposite number in the DNA strand being transcribed C. DNA helix reforms behind polymerase after it passes a particular stretch of DNA; only a few (~9) RNA nucleotides stay attached to DNA as DNA-RNA hybrid just behind site of polymerase operation D. Bacterial RNA polymerase can incorporate 50 - 100 nucleotides/sec; most genes in cell are transcribed simultaneously by numerous polymerases IV. Enzyme must stay attached to DNA over long stretches of template to make the prodigiously long mRNAs that they do, but be loose enough to move; the enzyme is processive Transcription: Prokaryotes I. E. coli has a single type of RNA polymerase - 5 subunits tightly associated to form core enzyme A. Purify core enzyme, add to solution of bacterial DNA & ribonucleoside triphophates —> enzyme binds to DNA & synthesizes RNA (but not the same RNA as in cell)
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1. It starts synthesis at inappropriate sites (random sites it would normally have ignored
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ch11e-mail (2) - CHAPTER 11 GENE EXPRESSION: FROM...

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