Questions and Answers
What is the function of the σ factor in RNA polymerase?
What is the purpose of genetic control of enzyme activity?
What is the function of repressors in genetic control?
What is the difference between an operon and a regulon?
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What is the function of activators in genetic control?
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What is the sequence of the promoter recognised by the classical 'vegetative' E. coli sigma factor?
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What is the function of translational control in genetic control?
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What is the purpose of the activator-binding site in genetic control?
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What is the role of lactose in the lactose operon?
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What happens to the repressor protein when lactose is present?
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What is the result of the repressor protein binding to the operator region?
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What is the function of the regulator gene in the lactose operon?
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What happens to the three enzyme genes in the absence of lactose?
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What is the role of the operator region in the lactose operon?
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What is the outcome when the inactivated repressor protein is unable to bind to the operator region?
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What type of protein is the repressor protein?
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What is the result of competitive inhibition of enzyme activity?
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What is the purpose of DNA ligase in gene cloning?
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What is the advantage of using plasmids as vectors?
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What is the function of restriction endonucleases?
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What is the objective of creating a DNA library?
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What is the purpose of PCR?
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What is a characteristic of plasmids?
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What is the purpose of using a four-cutter restriction enzyme in creating a DNA library?
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What is the result of the fourth step in gene cloning?
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What is the purpose of partial restriction endonuclease digestion?
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What is the purpose of size fractionation in DNA library creation?
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What is the type of end produced by restriction endonucleases?
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What is used to identify the presence of the target DNA?
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What is the purpose of cloning the cells that contain the target fragments?
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What is the result of a successful transformation reaction?
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What is the purpose of antibiotic selection in transformation?
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Why is it difficult to clone eukaryotes gene into prokaryote?
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How is mRNA extracted from other ribosomal RNA?
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What is the first step in converting mRNA into dsDNA?
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What is the function of the klenow fragment of E. coli DNA polymerase I?
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What is formed after reverse transcription of the mRNA?
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What is the purpose of treating the mRNA-bead complex with a buffer?
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Study Notes
Recombinant DNA Technology
Promoter Reminder
- Promoter sequences are recognized by RNA polymerase holoenzyme
- Promoter lies adjacent to an activator-binding site
- RNA polymerase is composed of 5 polypeptides (α2ββσ) when it binds to promoters
- σ factor is the protein that recognizes specific promoter sequences
- Most important nucleotides are centered at –35 and –10 relative to the transcription start-site for promoters recognized by the classical "vegetative" E. coli sigma factor, σ70
- Promoter sequence: 5'- TTGACA—17 bp spacing—TATAAT-5 to 9 bp- START Codon
Genetic Control
- Genetic control of enzyme activity refers to controlling transcription of mRNA needed for an enzyme's synthesis
- In prokaryotic cells, this involves induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase
- Regulatory proteins are part of either an operon or a regulon
- Operon: a set of genes transcribed as a polycistronic message that is collectively controlled by a regulatory protein
- Regulon: a set of related genes controlled by the same regulatory protein but transcribed as monocistronic units
- Regulatory proteins may function either as repressors or activators
Genetic Control Groups
- Repressors: Regulatory proteins that block transcription of mRNA
- Activators: Regulatory proteins that promote transcription of mRNA
- Translational control (antisense RNA): A strand of RNA complementary to a mRNA coding for some polypeptide or protein
Lactose Operon
- E. coli will only synthesize the three enzymes it requires to utilize lactose if that sugar is present in the surrounding environment
- Lactose functions as an inducer
- In the absence of lactose, the repressor protein binds to the operator and RNA polymerase is unable to transcribe the genes for utilization of lactose
- When lactose is present, it binds to the allosteric repressor protein and causes it to change shape, allowing RNA polymerase to transcribe the genes required for lactose degradation
Gene Cloning
- Gene cloning involves four steps:
- The vector DNA is cleaved with one or more restriction enzymes
- The DNA to be cloned is joined to the vector by ligase enzyme, generating a recombinant molecule
- The recombinant DNA is introduced into a host cell (e.g., bacteria)
- The transformed colony is selected to amplify
Vectors
- Vectors: DNA molecules used to transfer foreign DNA fragments between cells
- Types of vectors:
- Plasmid
- Phage (virus)
- Cosmid
- Yeast Artificial Chromosome (YAC)
- Bacterial Artificial Chromosome (BAC)
Creating and Screening a Library
- Isolation of genes encoding proteins is a fundamental objective of molecular biotechnology
- A DNA library for the selected organism can be generated by:
- Using restriction enzyme to cut the DNA into smaller fragments
- Inserting each fragment into a vector
- Cloning and identifying the cells containing the target fragments
- The presence of the target DNA can be identified with a DNA probe
Cloning DNA Sequence That Encode Eukaryotes Protein
- Due to post-translational modification and the presence of introns, it is difficult to clone eukaryotic genes into prokaryotic ones
- To solve this problem, mRNA can be extracted and used as all introns regions are already removed
- mRNA can be isolated using a special isolation technique based on devising a chromatography method where the beads are attached to poly TTTTT tail, which will bind to the poly AAAAA tail present at the end of the mRNA
- Cloning DNA sequence that encodes eukaryotic protein involves:
- Converting mRNA into dsDNA using reverse transcription and DNA synthesis
- Cloning the cDNA
- Identifying the transformed cell
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Description
This quiz covers the basics of recombinant DNA technology, including promoter sequences, RNA polymerase, and activator-binding sites. It's based on Lecture 5 of Microbial Biotechnology.