lecture 1: Prokaryotic transcription flashcards

The major advances in DNA technology are made possible by bacterial genetics. This includes understanding how prokaryotic organisms, specifically bacteria, manage gene regulation, the functionality of plasmids, and the applications of cloning.

Structure and Organization
- The prokaryotic genome is predominantly circular in shape.
- Genes within prokaryotes typically lack introns, which are non-coding sections of DNA found in eukaryotes.
- Genes are mostly organized in operons, allowing for closely regulated gene groups to be expressed simultaneously.
- Operon Definition: A unit made up of linked genes that is thought to regulate other genes responsible for protein synthesis.
- mRNAs produced are polycistronic, meaning they can encode multiple proteins within a single mRNA molecule.
- The prokaryotic genome is arranged less compactly than eukaryotic chromosomes.
- In bacteria, transcription and translation occur simultaneously, a process known as coupled transcription and translation.
- Bacteria also contain plasmids, small, extrachromosomal circular DNA that can replicate independently of chromosomal DNA.

Physical Map of E. coli
- Contains 4140 protein-coding genes and 178 RNA genes.
- Genome length is approximately 4,639,221 base pairs (bp).

Nucleotide Composition
- Purine nucleotides include Adenine (A) and Guanine (G). Their structures consist of a phosphate group, ribose sugar, and nitrogenous bases.
- Pyrimidine nucleotides include Cytosine (C) and Thymine (T).
- Example: Deoxyadenosine 5'-monophosphate (dAMP) is made of a phosphate group, ribose with a hydroxyl (-OH) group at 2', and the nitrogenous base adenine.
- Structures provided for Deoxyguanosine (dGMP), Deoxycytidine (dCMP), and Deoxythymidine (dTMP).

Processes Involved
- Replication: Duplication of DNA to pass on genetic information.
- Transcription: Process of copying a segment of DNA into RNA.
- Translation: Process of translating the RNA into a protein.
All processes proceed in the 5' to 3' direction.

Promoter Sequences
- Transcription initiation occurs downstream of promoter sequences in prokaryotes such as E. coli.
- Strong E. coli promoters have consensus sequences located at -35 and -10 regions relative to the transcription start site (+1).
Transcription Initiation
- RNA polymerase binds at specific promoter sequences, facilitating the transcription process. Important regions include the -35 and -10 sites.
RNA Polymerase Function
- The enzyme that synthesizes RNA from the DNA template during transcription.

Regulatory Proteins
- Transcription is regulated by activators and repressors.
- Positive Regulation: Activators enhance RNA polymerase activity, promoting transcription.
- Negative Regulation: Repressors inhibit RNA polymerase recruitment, reducing transcription.
Mechanisms of DNA bending and looping are employed by activators to enhance contact between DNA and RNA polymerase.

Functionality
- The lac operon is a classic example of gene regulation in prokaryotes, specifically E. coli. It is only transcribed in the presence of lactose.
- In the absence of lactose, the repressor protein blocks transcription by binding to the operator region.
- When lactose is present, it binds to the repressor, causing a conformational change that releases it from the operator, allowing transcription.
Gene Products
- The lac operon encodes three proteins: Beta-galactosidase (lacZ), Beta-galactoside permease (lacY), and Beta-galactoside transacetylase (lacA).

Plasmid Characteristics
- Plasmids can replicate independently of the bacterial chromosome and exist in either low-copy or high-copy number.
- Common features of plasmids include antibiotic resistance genes, such as for Ampicillin (AmpR).
Recombinant DNA Technology
- Involves joining together different DNA segments, typically using restriction enzymes that cut DNA at specific sites.
- This allows for the insertion of genes into plasmids for subsequent expression.

Mechanism
- Restriction enzymes recognize specific palindromic sequences in DNA and cleave it, creating sticky or blunt ends for ligation.
- Different enzymes exhibit variation in recognition sequences. Examples include EcoRI (GAATTC) and BamHI (GGATCC).

Process
- Transformation refers to the uptake of plasmids by bacteria, which can provide resistance to antibiotics.
- Successful transformation can be assessed through colony growth on selective medium (e.g., Amp or Kan).

Expression Systems
- Proteins can be synthesized in bacteria using vectors containing T7, T3, or SP6 promoters.
- The pET system is designed for expressing proteins with a His-Tag for purification via affinity chromatography.
Induction of Protein Production
- IPTG is often used to induce transcription in bacterial cells expressing T7 RNA polymerase, facilitating the production of proteins of interest.
- Analysis of protein expression can be performed via methods such as SDS-PAGE to assess yield and purity.

Coupled Transcription and Translation
- Allows for the synthesis of proteins in vitro using rabbit reticulocyte lysates containing necessary components (ribosomes, tRNAs, and amino acids).
- Restriction enzymes are used to prepare DNA templates for in vitro transcription.

Glutathione S-transferase (GST) fusion proteins can be affinity purified based on the interaction between GST and its substrate, glutathione.
The purification process is enhanced using beads that are coupled with glutathione, allowing the easy separation of GST-tagged proteins from the lysate.