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Lecture 2

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Biochemistry 2280A
Christopher Brandl

Transcription Bacterial RNApolymerase ● The enzyme that makes RNAtranscripts using DNAas a template and nucleoside triphosphates (NTPs) as substrates ○ the key molecules that make RNAs from DNA! ● It is a multi-subunit enzyme; 4 subunits: two alpha subunits, a beta subunit, and a beta prime subunit ● The core enzyme does not recognize promoters; rather the promoter specificity of RNA polymerase is determined by the sigma subunit ○ Enzyme + sigma subunit = RNApolymerase holoenzyme ● The sigma subunit recognizes and contacts the -10 and -35 sequences; makes base contact required for recognition ● Sigma subunits are one of the many examples demonstrating the importance of protein- nucleic acid interactions Steps in Initiation of Transcription 1. RNAholoenzyme binds the promoter; once bound is called thd closed complex 2. RNApolymerase unwinds the DNAstrands, while still bound, around the start site -- open complex (after it unwinds the strands) 3. The first NTP (not a dNTP) is brought to the template -- no primer is required 4. Using NTPs as substrates, chain elongation proceeds in a 5’-3’direction, following base pairing rules; RNA synthesis begins a. in this process, phosphodiester bonds form and pyrophosphate is released 5. After the addition of 5-10 nucleotides, sigma falls off the holoenzyme; this signals the enzyme to race down the DNAallowing the rest of transcription to occur 6. The transcription bubble moves downstream with the template DNAreannealing behind 7. Chain elongation proceeds until a terminator is reached and RNAPolymerase falls off 8. Sigma rebinds RNApolymerase and the cycle is repeated Regulation of Gene Expression ● How are different bacterial promoters transcribed at different levels? What causes those differences? ● One reason is that some genes have better (more attractive) -10 and -35 sequences ○ the closer the sequences are closer to the consensus sequences, the better and higher levels of expression ○ this is not a dynamic regulation as the genes cannot change their -10 and -35 sequences; it is fixed ● Also, there can be more than one sigma factor ○ each sigma factor recognizes different promoter sequences ○ allows genes to be expressed at different times and different levels ● There are many gene specific regulatory proteins (is dynamic) ○ negative regulation: factors repress transcription ○ positive regulation: factors activate transcription Negative Regulation ● Trp Operon requires 5 genes to synthesize tryptophan and are transcribed from a common promoter ● The trp operon is expressed when there is little tryptophan in the cellular environment therefore the operon is regulated by tryptophan itself ● Between the -10 and -35 region of the trp promoter, there is a DNAsequence called the trp operator that binds a protein called the trp repressor ● At high concentration of tryptophan, trp repressor binds tryptophan ● The trp repressor-tryptophan complex binds the trp operator DNA ● The trp repressor-tryptophan complex blocks RNApolymerase from the promoter by blocking the -10 and -35 regions on the promoter needed for beginning expression ● When tryptophan concentration is low, tryptophan dissociates from the trp repressor ● The trp repressor no longer binds the trp operator, RNApolymerase binds the promoter and transcription results ● To summarize, low tryptophan concentration leads to the gene being expressed, high concentration of tryptophan leads to gene expression being blocked What is the Structural Basis for Tryptophan? ● Trp Repressor is not very long (107 amino acids) and consists of mainly alpha helices ● However, alpha helices 4 and 5 make up the helix-turn-helix motif which is important for ● The trp repressor acts as a dimer and has a 2-fold symmetry held together by contact via the alpha 3 helices of each monomer ● The two helix 5s recognize adjacent major grooves in the operator DNA; the minor groove is not large enough to fit the alpha helix o Most alpha helices therefore interact with major grooves as they are just large enough ● When tryptophan binds to the trp promoter, it induces a conformational change in the trp repressor which allows DNAbinding resulting in the helix 5’s tilting inward and not being able to recognize and interact with the major grooves of the DNA ● General themes to take fro
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