PCR, Replication And Transcription, And Lac Operon Questions

1.) PCR a.) Using the dsDNA sequence 1 as a template, you want to generate dsDNA sequence 2 using PCR. What are the sequences of the two primers you use? 15nt of each primer have to anneal perfectly to the template. Write the primer sequences in 5’->3’ direction in the provided space. (25 points) Sequence 1 5-TATAGGACGATGTTGATGAATGGTACAATCACAGTACGTACGTACAGTCAGTGAAA-3 Sequence 2 5-TAGCGGTACATGTTGATGAATGGTACAATCACAGTACGTACGTACAGTCTATCGAT-3 Primer 1 5- -3 Primer 2 5- -3 b.) You amplify a double-stranded DNA template using PCR. Draw a schematic of the first 2 rounds of the reaction (Each round has 3 steps: 1. Denature, 2. Anneal, and 3. Extend) (25 points) 2.) Replication and Transcription a) Draw a DNA Replication fork. Label all features, enzymes, and molecules we discussed in class. Describe the function of each protein. (10 words per enzyme) (25 points) b) Draw an elongating RNA polymerase. Label all features, enzymes, and molecules we discussed in class. Describe the different functions RNA polymerase is performing (10 words per function) (25 points) 3.) Bacterial Transcriptional Regulation: Lac Operon a) Draw the lac operon promoter region with all its regulators (and polymerase if appropriate) in the presence of lactose and the presence of glucose in the growth media. Indicate the level of transcription from the lac operon under those conditions (no expression, low expression, or high expression). (25 points) b) Draw the lac operon promoter region with all its regulators (and polymerase if appropriate) in the presence of lactose and the presence of glucose in the growth media in bacteria with a mutation in the CAP protein that renders it constitutively active even in the absence of cAMP. Indicate the level of transcription from the lac operon under those conditions (no expression, low expression, or high expression). (25 points) 4.) Transcription a)True/False (2 points each): _____ RNA polymerase copies both strands of DNA during transcription. _____ RNA polymerase starts transcription at the Start Codon and stops at the Stop Codon. _____ Bacterial RNA polymerase requires �-factor to effectively initiate transcription. _____ RNA polymerase generates Okazaki Fragments on the Lagging strand. _____ RNA polymerase copies the entire genome before every cell division. b) Directionality: RNA polymerase initiates transcription of the double stranded DNA given below at the indicated transcription start site. Write the sequence of the resulting RNA in the 5’ to 3’ direction into the box given below. (10 points) 5.) DNAseI Footprinting You perform 4 DNAseI footprinting experiments. All the experiments contain DNA 1. Experiments 2,3, and 4 also contain Protein A which binds DNA 1 in the indicated position. DNA 1 is labeled by radio labels on the indicated ends. Arrows show positions that DNAseI can cut. Draw the banding pattern you expect to see in the 4 different experiments in the box on the right. (30 points)