A. In prokaryotes, transcription and translation steps are coupled. 2 B. An leader RNa is transcribed before the first structural gene trpE; this leader RNa contains an open reading frame which encodes a leader peptide and RNa elements 1, 2, 3, 4. Regions 3, 4 can form a hairpin which is followed by 8 US. This is a characteristic transcription terminator. Region 2 and 3 can also form a more stable hairpin, and this hairpin formation excludes the terminator structure C. In the region 1 of the leader RNA, two adjacent tryptophan codons are present. When the tryptophan concentration is high, the ribosome proceeds through region l to region 2, resulting in the hairpin structure with regions 3 and 4 being paired and thus the transcription is terminated When the tryptophan concentration is low, the ribosome is caught in the region 1, resulting regions 2 and 3 paired hairpin, which inhibits the terminator formation. In this case, the rnaP can pass the premature terminator and transcribes trp genes 5. Please list the different DNa binding domains and activating regions of the eukaryotic transcription activators. (10 points) DNa binding domains include: Homeodomain proteins containing the helix-turn-helix dna binding motif, Zinc containing DNA-binding domains (Zinc finger domain and Zinc cluster domain); leucine zipper motifs and helix-loop-helix(HLH) proteins Activating regions are not well-defined structures. They are grouped on the basis of amino acid content. Some main groups include acidic activating region, glutamine-rich activating region, and proline-rich activating region 6. Yeast two-hybrid method is developed from the basic structural feature of a well-studied transcription activator: can you describe how yeast two-hybrid method works to study protein-protein interaction?(10 points) Yeast two-hybrid method is based on the fact that the eukaryotic activator GaL has separate dna binding region and activating region. This method is used to identify and study the protein-protein interaction Assay(taking Protein a b as an example (1) Fuse the genes encoding protein A(bait) and B(prey) to the dnA fragments encoding the dna binding domain and activating domain, respectively 2)Express two fused proteins in the same yeast cell clo 3)In the yeast cell, fused protein A will bind to the promoter of the report gene. If there is no interaction between protein A and B, the report gene cannot express. If there is interaction between protein A and B, the activating region-B-A-DNA binding domain complex will form, and the activating region will therefore recruit RNa Polymerase to initiate the transcription of the downstream report gene. In sum, the expression of report gene indicates that there is a protein-protein interaction between A and BA. In prokaryotes, transcription and translation steps are coupled. 2’ B. An leader RNA is transcribed before the first structural gene trpE; this leader RNA contains an open reading frame which encodes a leader peptide and RNA elements 1, 2, 3, 4. Regions 3, 4 can form a hairpin, which is followed by 8 Us. This is a characteristic transcription terminator. Region 2 and 3 can also form a more stable hairpin, and this hairpin formation excludes the terminator structure. 3’ C. In the region 1 of the leader RNA, two adjacent tryptophan codons are present. When the tryptophan concentration is high, the ribosome proceeds through region 1 to region 2, resulting in the hairpin structure with regions 3 and 4 being paired and thus the transcription is terminated. When the tryptophan concentration is low, the ribosome is caught in the region 1, resulting regions 2 and 3 paired hairpin, which inhibits the terminator formation. In this case, the RNAP can pass the premature terminator and transcribes trp genes. 3’ 5. Please list the different DNA binding domains and activating regions of the eukaryotic transcription activators. (10 points) DNA binding domains include: Homeodomain proteins containing the helix-turn-helix DNA binding motif; Zinc containing DNA-binding domains (Zinc finger domain and Zinc cluster domain); leucine zipper motifs and helix-loop-helix (HLH) proteins 6’ Activating regions are not well-defined structures. They are grouped on the basis of amino acid content. Some main groups include acidic activating region, glutamine-rich activating region, and proline-rich activating region. 4’ 6. Yeast two-hybrid method is developed from the basic structural feature of a well-studied transcription activator; can you describe how yeast two-hybrid method works to study protein-protein interaction? (10 points) Yeast two-hybrid method is based on the fact that the eukaryotic activator GAL4 has separate DNA binding region and activating region. This method is used to identify and study the protein-protein interaction. 2’ Assay (taking Protein A & B as an example): (1) Fuse the genes encoding protein A (bait) and B (prey) to the DNA fragments encoding the DNA binding domain and activating domain, respectively. 3’ (2) Express two fused proteins in the same yeast cell clone. 1’ (3) In the yeast cell, fused protein A will bind to the promoter of the report gene. If there is no interaction between protein A and B, the report gene cannot express. If there is interaction between protein A and B, the activating region-B-A-DNA binding domain complex will form, and the activating region will therefore recruit RNA Polymerase to initiate the transcription of the downstream report gene. In sum, the expression of report gene indicates that there is a protein-protein interaction between A and B. 4’