Chap 7 Problem 1 In glucose media without

Chap. 7 Problem 1 In glucose media without lactose, the lac repressor is bound to the lac operator, and the CAP protein is not bound to its control site near the promoter due to low c. AMP level. As a result, transcription of the lac operon is shut off. In lactose media lacking glucose, the operon is turned on and transcription occurs at the highest rate. c. AMP is synthesized in the absence of glucose, and the CAP-c. AMP complex binds to its control element stimulating transcription initiation by RNA polymerase. Due to binding of lactose to the lac repressor, the complex leaves the operator and transcription no longer is blocked by the repressor (Fig. 7. 3).

Chap. 7 Problem 3 * * * Part 1: Classes of RNA transcribed by RNA Pols I, II, and III, that are important to know, are marked with asterisks (Table 7. 2). Part 2: RNA polymerase II is very sensitive to inhibition by the Amanita phalloides poison called -amanitin. The activity of RNA Pol II, but not Pols I & III is inhibited at a 1 g/ml concentration. Therefore, one can determine if a particular gene is transcribed by RNA Pol II by determining if 1 g/ml -amanitin inhibits transcription of the gene.

Chap. 7 Problem 5 TATA boxes, Cp. G islands, and initiators all serve as promoters, which set the transcription start site for genes. The TATA box is found in genes that are strongly expressed, and therefore was the first promoter element to be identified by in vitro transcription assays. In addition, the transcription start site occurs at a fixed location downstream of a TATA box (Fig. 7. 14).

Chap. 7 Problem 6 A commonly used method to detect promoter-proximal control elements is linkerscanning mutagenesis (Fig. 7. 21). In this technique, a segment of random DNA is substituted for DNA sequences across the control region. Reporter gene assays are used to determine if the substitutions block transcription, indicating a control region is present.

Chap. 7 Problem 7 Promoter-proximal elements typically are functional only when close to the promoter, whereas distal enhancers often can function at variable distances from the promoter (Fig. 7. 22). Distal enhancers sometimes can be moved to the other side of the gene and still regulate transcription. Both types of sequences are bound by transcription activators that help RNA Pol II load onto promoters.

Chap. 7 Problem 8 In DNase I footprinting, DNA labeled on one strand is incubated with a transcription factor (TF), and the complex is treated with a small amount of DNase I, which cleaves DNA where it is not masked by the TF (Fig. 7. 23 a). A control DNA sample lacking the TF is treated under parallel conditions. The banding patterns from the two samples are compared by gel electrophoresis to locate the "footprint" region where the TF has shielded the DNA from cleavage. In gel-shift assays (electrophoretic mobility shift assays) (Fig. 7. 24), a labeled DNA fragment (200 -300 bp) containing the binding site is incubated with the TF and then is run on a polyacrylamide or agarose gel. A sample of the DNA lacking the protein is run in parallel. Bound DNA fragments run more slowly and are shifted to a higher position on the gel.

Chap. 7 Problem 9 Transcription factors have a modular structure consisting of at least two domains. Transcriptional activators contain a DNA binding domain and an activation domain. Transcriptional repressors contain a DNA binding domain and a repression domain. Some TFs also contain a ligand binding domain that regulates activity. Domains typically are joined together in a single polypeptide by flexible linker sequences that serve as hinges and allow conformational changes needed for activation/repression. Some examples of transcriptional activators are shown in Fig. 7. 27.

Chap. 7 Problem 12 The sequential assembly of Pol II transcription pre-initiation complex is shown in Fig. 7. 17. TBP (TATA boxbinding protein) binds first and determines where transcription will initiate. TFIIH is the last factor to bind. TFIIH has a helicase activity that is important in melting DNA and generating an open complex wherein the template strand is located within the active site of the polymerase. TFIIH also phosphorylates the CTD of Pol II making the enzyme highly processive.

Chap. 7 Problem 16 UASs are comparable to promoter-proximal elements and enhancers in higher eukaryotes (Fig. 7. 22).

Chap. 7 Problem 22 The four main classes of DNA-binding proteins we have discussed are the 1) helix-loop-helix proteins (Fig. 7. 28), 2) basic zipper (b. ZIP) proteins (Fig. 7. 29 c), 3) basic helix-loop-helix (b. HLH) proteins (Fig. 7. 29 d), and zinc-finger proteins (Fig. 7. 29 a & b). A detailed description of their structural features is presented in the text and lecture slides. Zinc finger TFs are the most common type of DNA binding protein encoded by the human genome.