Molecular iology Course-quiz 3 Chapter 16-18(90 min) December22,2006 Total 130 points Note: the answers should be concise and hit the points(答案要尽量简明和击中要 点) 1. What are the gene expression stages that can be regulated? (10 points) Regulated stages in gene expressio (1)transcription initiation(the most important stage): RNAP binding of the promoter, transition from the closed complex to the open complex; promoter (2)stages after initiation(elongation and termination ()RNA processing(alternative splicing)in eukaryotes 2 (4)translation 2. What is the recruitment regulation? Please describe an example of this kind of gene regulation in bacteria. 10 points Recruitment regulation: refers to the regulator protein bound to a dna egion can recruit the components in the transcription machinery or the nucleosome modifier, and thus exert an alteration of the transcription level. This class of regulator has a dNa binding domain and a protein interaction domain For example, in Lac operon of E. coli, the activator CAP binds to the CAP binding site and recruits the rna polymerase to bind its promoter efficiently for transcription initiation. (Other correct example will also do) 3. What is the allostery regulation? Please describe an example of this kind of gene regulation in bacteria. (10 points Allostery regulation: binding of the transcription activator induces a conformational change in the transcription complex (either polymerase or promoter DNA), which transforms the transcription complex from the inactive form to the active form, resulting in transcription activation. 5 Examples include activators NtrC and Merr, which work by induce onformational change in the polymerase and promoter dna respectively.(One example will suffice) 4. How the expression of a tryptophan operon is regulated? (15 points) The expression of tryptophan operon are controlled by two layers of regulation 1) At the transcription initiation stage: E. coli encodes a trp repressor, however the repressor alone cannot bind to the operator. Tryptophan acts as a corepressor of the trp operon. When the tryptophan level is high in bacteria, it binds to the trp repressor and the complex binds to the operator to repress the transcription initiation 2) Attenuation: a regulation by premature transcription termination within the leader rna
Molecular Biology Course- quiz 3 Chapter 16-18 (90 min) December 22, 2006 [Total 130 points] Note: the answers should be concise and hit the points (答案要尽量简明和击中要 点)。 1. What are the gene expression stages that can be regulated? (10 points) Regulated stages in gene expression: (1) transcription initiation (the most important stage): RNAP binding of the promoter, transition from the closed complex to the open complex; promoter escape 4’ (2) stages after initiation (elongation and termination) 2’ (3) RNA processing (alternative splicing) in eukaryotes 2’ (4) translation 2’ 2. What is the recruitment regulation? Please describe an example of this kind of gene regulation in bacteria. (10 points) Recruitment regulation: refers to the regulator protein bound to a DNA region can recruit the components in the transcription machinery or the nucleosome modifier, and thus exert an alteration of the transcription level. This class of regulator has a DNA binding domain and a protein interaction domain (region). 5’ For example, in Lac operon of E. coli, the activator CAP binds to the CAP binding site and recruits the RNA polymerase to bind its promoter efficiently for transcription initiation. (Other correct example will also do) 5’ 3. What is the allostery regulation? Please describe an example of this kind of gene regulation in bacteria. (10 points) Allostery regulation: binding of the transcription activator induces a conformational change in the transcription complex (either polymerase or promoter DNA), which transforms the transcription complex from the inactive form to the active form, resulting in transcription activation. 5’ Examples include activators NtrC and MerR, which work by induce conformational change in the polymerase and promoter DNA respectively. (One example will suffice) 5’ 4. How the expression of a tryptophan operon is regulated? (15 points) The expression of tryptophan operon are controlled by two layers of regulation: 1) At the transcription initiation stage: E. coli encodes a trp repressor, however the repressor alone cannot bind to the operator. Tryptophan acts as a corepressor of the trp operon. When the tryptophan level is high in bacteria, it binds to the trp repressor and the complex binds to the operator to repress the transcription initiation. 5’ 2) Attenuation: a regulation by premature transcription termination within the leader RNA. 2’
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 B
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. 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’
7. How the eukaryotic transcription activators and repressors regulate transcription itation?(10 points) Activators: (1)recruit the transcriptional machinery to the gene(2.5).(2)recruit nucleosome modifiers that help the transcription machinery bind at the promoter Repressors: activator's binding site, a repressor inhibit binding of the activator. 1 b. Inhibition--- A repressor binds to a site besides an activator occluding its activating region. c. Direct repression---A repressor binds to a site upstream of a gene, by interacting with the transcriptional machinery at the promotor in some way, inhibits transcriptional inhibition d. Indirect repression--- recruit nucleosome modifiers that alter nucleosomes to inhibit transcription. This is the most common mechanism of the repressor inhibition of the transcription in eukaryotes 8. Please describe how the transcription activators Sw15 and SBF work synergistically to have the yeast Ho gene expressed only in mother cells and at certain point in the cell cycle, resulting in the budding division feature of yeast S cerevisiae.(10 points SW15: acts only in the mother cell(1)and binds to multiple sites unaided some distance from the promoter of the HO gene(1) SBF: acts only at the correct stage of the cell cycle(1)and cannot bind its sites unaided near to the promoter of the HO gene(1), Thus, when both activators are present and active, the action of sw15 opens the multiple binding sites of SBF and enables SBF to bind. SBF, in turn, recruits the transcriptional machinery by directly binding Mediator and activates expression of the HO gene 5 Therefore, through the cell-type specific activation of sw15 and the cell cycle-specific activation of SBF, and the synergistic interaction of these two activators, the yeast HO gene expressed only in mother cells and at certain point in the cell cycle 9. What is RNA interference, and what are the molecular mechanisms that lead to the observed phenotype of RNA interference? (10 points) RNA interference (RNAi) refers to a phenomenon that double-stranded RNA inhibits expression of genes homologous to that dsRna Mechanism of RNAi: the dsRNa is first chopped to siRna by dicer(2), and the SiRNA is incorporated into the effector complex called RISC(RNA interference silencing complex)(2 ). Then the siRISC complex will find its homologous mRNAs to cause the degradation or translational inhibition of the target mRNAs (2). The siRISC complex can also interact with its homologous DNA sequence in the promoter
7. How the eukaryotic transcription activators and repressors regulate transcription initiation? (10 points) Activators: (1) recruit the transcriptional machinery to the gene (2.5’). (2) recruit nucleosome modifiers that help the transcription machinery bind at the promoter (2.5’). Repressors: a. Competition--- By binding to a site on DNA that overlaps the activator’s binding site, a repressor inhibit binding of the activator. 1’ b. Inhibition--- A repressor binds to a site besides an activator, occluding its activating region. 1’ c. Direct repression--- A repressor binds to a site upstream of a gene, by interacting with the transcriptional machinery at the promotor in some way, inhibits transcriptional inhibition. 1’ d. Indirect repression--- recruit nucleosome modifiers that alter nucleosomes to inhibit transcription. This is the most common mechanism of the repressor inhibition of the transcription in eukaryotes 2’ 8. Please describe how the transcription activators SWI5 and SBF work synergistically to have the yeast HO gene expressed only in mother cells and at certain point in the cell cycle, resulting in the budding division feature of yeast S. cerevisiae. (10 points) SWI5: acts only in the mother cell (1’) and binds to multiple sites unaided some distance from the promoter of the HO gene (1’). SBF: acts only at the correct stage of the cell cycle (1’) and cannot bind its sites unaided near to the promoter of the HO gene (1’), Thus, when both activators are present and active, the action of SWI5 opens the multiple binding sites of SBF and enables SBF to bind. SBF, in turn, recruits the transcriptional machinery by directly binding Mediator and activates expression of the HO gene. 5’ Therefore, through the cell-type specific activation of SWI5 and the cell cycle-specific activation of SBF, and the synergistic interaction of these two activators, the yeast HO gene expressed only in mother cells and at certain point in the cell cycle. 1’ 9. What is RNA interference, and what are the molecular mechanisms that lead to the observed phenotype of RNA interference? (10 points) RNA interference (RNAi) refers to a phenomenon that double-stranded RNA inhibits expression of genes homologous to that dsRNA. 3’ Mechanism of RNAi: the dsRNA is first chopped to siRNA by dicer (2’), and the siRNA is incorporated into the effector complex called RISC (RNA interference silencing complex) (2’). Then the siRISC complex will find its homologous mRNAs to cause the degradation or translational inhibition of the target mRNAs (2’). The siRISC complex can also interact with its homologous DNA sequence in the promoter
egion; then an action of the chromatin remodeling, it can cause the inhibition on the transcription of the target genes(1) 10. How miRNAs are generated in eukaryotic cells, and how they regulate gene expression? Please list some biological function of miRNAs. (10 points) The miRNA is generated through the pathway described (1)Primary miRNA(Pri-miRNA) is transcribed from the genome of the eukaryotic cells, independently as a mirNa gene or as a part of a mRNA gene in the non-coding region. The pri-miRNA is usually 5'capped and has 3 poly-A tail (2)Pri-miRNA is then processed by Drosha in nucleus, which removes its 5 and 3 end to produce the pre-miRNA(precursor mIRNA) (3)Pre-miRNA is then transported to cytoplasm, where Dicer further process it to the short double helical miRNA of about 21-23 bp The miRNA can regulate gene expression by causing the degradation and/or translational inhibit ion of the homologous mRNAs Biological functions. MiRNAS are involved in animal development, phenotype determination of plants; the differentiation of stem cells, cancer development and probably viral infections. 3(two functions are required) The methods used to produce siRNAs:(nc飞么 11. Do you know how to produce siRNAs? What are ations bile 1)Chemical synthesis 2)In vitro transcription of pre-miRNA with T7 promote 3)In vitro transcription of long dsRna by that is then cleaved by E. coli rNase III or rNase lll-like dicer 4) Transcribed by RNaP Ill in living cells from the transfected DNA plasmid driven by a RNAP Ill promoter such as U6 or HI Applications:(7 points) of th target genes bearing homologous sequence in vivo can be widely applied in biolog ical research and developing new therapeutic treatments the Biological research: siRNA provides a power ful reverse genetics method to study the biological function of an interested gene SIRNA also can be used to perform genome-wide screening of new genes involving in a biological function. Developing new therapeuticS: SiRNA can be used to knock-down the expression of genes from viral pathogens, and of oncogenes causing cancer or other disease-causing genes. Therefore, siRNA provides a powerful approach to combat viral infection and genetic diseases 12. How is the sex determination in fly controlled by the regulation of transcription initiation and alternative splicing?(15 points) A. Early transcriptional regulation of sxl in maile and female flies
region; then an action of the chromatin remodeling, it can cause the inhibition on the transcription of the target genes (1’). 10. How miRNAs are generated in eukaryotic cells, and how they regulate gene expression? Please list some biological function of miRNAs. (10 points) The miRNA is generated through the pathway described: (1) Primary miRNA (Pri-miRNA) is transcribed from the genome of the eukaryotic cells, independently as a miRNA gene or as a part of a mRNA gene in the non-coding region. The pri-miRNA is usually 5’capped and has a 3’ poly-A tail. 2’ (2) Pri-miRNA is then processed by Drosha in nucleus, which removes its 5’ and 3’ end to produce the pre-miRNA (precursor miRNA). 1’ (3) Pre-miRNA is then transported to cytoplasm, where Dicer further process it to the short double helical miRNA of about 21-23 bp 2’ The miRNA can regulate gene expression by causing the degradation and/or translational inhibition of the homologous mRNAs. 2’ Biological functions. MiRNAs are involved in animal development, phenotype determination of plants; the differentiation of stem cells, cancer development and probably viral infections. 3’ (two functions are required) 11. Do you know how to produce siRNAs? What are the applications of siRNAs in biological research and developing new therapeutics? (10 points) The methods used to produce siRNAs: (3 points) 1) Chemical synthesis 1’ 2) In vitro transcription of pre-miRNA with T7 promoter. 1’ 3) In vitro transcription of long dsRNA by that is then cleaved by E. coli RNase III or RNase III-like Dicer. 1’ 4) Transcribed by RNAP III in living cells from the transfected DNA plasmid driven by a RNAP III promoter such as U6 or H1. 1’ Applications: (7 points) The capability of siRNA in specifically down-regulating the expression of the target genes bearing homologous sequence in vivo can be widely applied in biological research and developing new therapeutic treatments. 1’ Biological research: siRNA provides a powerful reverse genetics method to study the biological function of an interested gene. siRNA also can be used to perform genome-wide screening of new genes involving in a biological function. 3’ Developing new therapeutics: siRNA can be used to knock-down the expression of genes from viral pathogens, and of oncogenes causing cancer or other disease-causing genes. Therefore, siRNA provides a powerful approach to combat viral infection and genetic diseases. 3’ 12. How is the sex determination in fly controlled by the regulation of transcription initiation and alternative splicing? (15 points) A. Early transcriptional regulation of Sxl in maile and female flies:
The Sis A and Sis b genes are found on the X cromosome and encode transcriptional activators that control expression of the Sxl gene. Dpn, a repressor of Sxl, is encoded by a gene on an autosome. The ratio of activators to repressors differs in the two sexes, and the 2: 1 ratio between the activators and repressors in female activates the transcription of Sxl gene(sex-lethal gene) from the Pe promoter. The translational product is called early Sxl protein B Regulation of the alternative splicing of sex-determinant mRNAs leads to different sexes. In females. the early Sxl protein acts as the splicing repressor to ensure the production of the mature Sxl mRNA transcribed from another promoter called Pm, leading to the functional Sxl protein(2). Sxl protein also acts as the splicing repressor of Tra pre-mRNA. Leading to the production of the functional Tra protein(2). Tra protein, together with the Tra-2, acts as the splicing activator for dsx double sex) pre-mRNA Splicing, resulting in the production of one isoform of DsX protein that represses male gene expression and activated female gene expression(2) As a result, female development is established. In males, without Sxl proteins Pre-mRNA of Sxl gene from the Pm promoter and tra gene could not be spliced to produce functional Sxl protein nor the Tra protein. Thus, pre-mRNA of dsx gene is spliced in another way to give a dsx protein isoform that represses female genes Therefore, male development is established(3) Score plot The average score is 97.35 9分--9-9
The Sis A and Sis B genes are found on the X cromosome and encode transcriptional activators that control expression of the Sxl gene. Dpn, a repressor of Sxl, is encoded by a gene on an autosome. The ratio of activators to repressors differs in the two sexes, and the 2:1 ratio between the activators and repressors in female activates the transcription of Sxl gene (sex-lethal gene) from the Pe promoter. The translational product is called early Sxl protein. 6’ B. Regulation of the alternative splicing of sex-determinant mRNAs leads to different sexes. In females, the early Sxl protein acts as the splicing repressor to ensure the production of the mature Sxl mRNA transcribed from another promoter called Pm, leading to the functional Sxl protein (2’). Sxl protein also acts as the splicing repressor of Tra pre-mRNA. Leading to the production of the functional Tra protein (2’). Tra protein, together with the Tra-2, acts as the splicing activator for dsx (double sex) pre-mRNA splicing, resulting in the production of one isoform of Dsx protein that represses male gene expression and activated female gene expression (2’). As a result, female development is established. In males, without Sxl proteins, Pre-mRNA of Sxl gene from the Pm promoter and tra gene could not be spliced to produce functional Sxl protein nor the Tra protein. Thus, pre-mRNA of dsx gene is spliced in another way to give a Dsx protein isoform that represses female genes. Therefore, male development is established (3’). 0 5 10 15 20 25 <30 30 ~50 50 ~70 70 ~80 80 ~90 90 ~100 100 ~110 110 ~120 120 ~130 Score Plot The average score is 97.35
