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Semester 2: Molecular Biology and Genetic Engineering
Gene organization and regulation of gene expression: Structure of DNA, Types of DNA, Gene organization in prokaryotes and eukaryotes, polycistronic genes, split genes promoters, enhancers, Regulation of gene expression: lac and trp operons in E. coli
Gene organization and regulation of gene expression
Structure of DNA
DNA is composed of two strands forming a double helix, where each strand is made up of nucleotides containing a phosphate group, a sugar, and a nitrogenous base. The four bases are adenine, thymine, cytosine, and guanine. The structure allows DNA to replicate and store genetic information.
Types of DNA
There are different types of DNA including chromosomal DNA, mitochondrial DNA, and plasmid DNA. Chromosomal DNA is found in the nucleus of eukaryotic cells, while mitochondrial DNA is located in mitochondria. Plasmid DNA is a small circular DNA molecule found in prokaryotes.
Gene organization in prokaryotes
In prokaryotes, genes are often organized in operons, which are groups of genes transcribed together under a single promoter. This arrangement allows for coordinated expression of related genes.
Gene organization in eukaryotes
Eukaryotic genes are usually organized individually and can have introns and exons. Introns are non-coding regions that are spliced out during RNA processing, while exons are coding regions that remain.
Polycistronic genes
Polycistronic genes are operons containing multiple coding sequences that can be transcribed together into a single mRNA molecule. This is common in prokaryotes.
Split genes
Split genes are those containing both introns and exons. The presence of introns is a characteristic feature of many eukaryotic genes, and they are removed during the post-transcriptional modification of RNA.
Promoters
Promoters are DNA sequences located at the beginning of a gene and are essential for the initiation of transcription. They provide a binding site for RNA polymerase and transcription factors.
Enhancers
Enhancers are regulatory DNA sequences that can enhance the transcription of associated genes. They can function at a distance from the promoter and are bound by specific proteins that facilitate transcription.
Regulation of gene expression
Gene expression can be regulated at various levels including transcriptional, post-transcriptional, translational, and post-translational levels.
Lac operon in E. coli
The lac operon is a model for gene regulation. It consists of genes required for lactose metabolism. In the presence of lactose, the lac repressor is inactivated, allowing transcription.
Trp operon in E. coli
The trp operon regulates tryptophan biosynthesis. When tryptophan is abundant, the operon is repressed, preventing unnecessary production of the amino acid.
DNA Replication and DNA polymerases: Replication in prokaryotes and eukaryotes, initiation at replication origins, Structure and function of DNA polymerases
DNA Replication and DNA polymerases
Overview of DNA Replication
DNA replication is the process by which a cell duplicates its DNA before cell division. It ensures that each daughter cell receives an exact copy of the genetic material. The process involves unwinding the double helix structure of DNA and synthesizing new complementary strands.
Replication in Prokaryotes
In prokaryotes, DNA replication begins at a specific region known as the origin of replication. The circular DNA molecule is unwound by helicases, and replication occurs bidirectionally. DNA polymerase III is the primary enzyme responsible for synthesizing new DNA strands, and primase lays down RNA primers to initiate synthesis.
Replication in Eukaryotes
Eukaryotic DNA replication is more complex due to the linear structure of chromosomes and a larger amount of DNA. It occurs at multiple sites along the DNA, with origins of replication being recognized by origin recognition complexes. DNA polymerase alpha starts the synthesis of new strands, followed by DNA polymerases delta and epsilon for elongation.
Initiation at Replication Origins
Replication origins are specific sequences in the DNA where replication begins. In eukaryotes, the assembly of pre-replication complexes is crucial for initiating replication. Key proteins such as the minichromosome maintenance (MCM) complex help in helicase activity and unwinding DNA.
Structure of DNA Polymerases
DNA polymerases are multi-subunit enzymes with distinct domains responsible for different functions. They include an active site for nucleotide incorporation, an exonuclease site for proofreading, and a clamp that increases processivity during DNA synthesis.
Function of DNA Polymerases
DNA polymerases play vital roles in DNA replication, repair, and recombination. They synthesize new DNA strands by adding nucleotides complementary to the template strand. The proofreading ability of some polymerases enhances the fidelity of DNA replication.
Transcription and mRNA processing: RNA structure and types, transcription mechanisms in prokaryotes and eukaryotes, transcription factors, RNA polymerases, initiation, elongation and termination, RNA processing (splicing, capping, polyadenylation)
Transcription and mRNA processing
RNA structure and types
RNA is a nucleic acid composed of ribonucleotides. The main types of RNA include messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and other non-coding RNAs. mRNA carries genetic information from DNA to the ribosome for protein synthesis. tRNA brings amino acids to the ribosome during translation, while rRNA is a structural component of ribosomes.
Transcription mechanisms in prokaryotes
In prokaryotes, transcription occurs in the cytoplasm. RNA polymerase binds to the promoter region of a gene to initiate transcription. There is no pre-mRNA; the mature mRNA is synthesized directly. Termination can occur through intrinsic termination, where a sequence forms a hairpin loop, or extrinsic termination, requiring a specific protein.
Transcription mechanisms in eukaryotes
In eukaryotes, transcription occurs in the nucleus. RNA polymerase II is responsible for mRNA synthesis. It requires a series of transcription factors to bind to the promoter. Transcription elongates as RNA polymerase moves along the DNA strand, and termination is regulated by specific sequences.
Transcription factors
Transcription factors are proteins that help regulate gene expression by binding to specific DNA sequences. They assist RNA polymerase in binding to the promoter and can act as activators or repressors, influencing the rate of transcription.
RNA polymerases
Eukaryotes have three main types of RNA polymerases: RNA polymerase I (rRNA), RNA polymerase II (mRNA), and RNA polymerase III (tRNA and other small RNAs). Prokaryotes have a single RNA polymerase that synthesizes all types of RNA.
Initiation, elongation, and termination in transcription
In initiation, RNA polymerase binds to the promoter with the help of transcription factors. Elongation involves the addition of ribonucleotides complementary to the DNA template. Termination signals the end of transcription, involving specific sequences or additional factors.
RNA processing: splicing, capping, and polyadenylation
After transcription, eukaryotic mRNA undergoes processing. Splicing removes introns and joins exons, capping adds a 5' cap for stability and recognition, and polyadenylation adds a poly-A tail to the 3' end, enhancing stability and export from the nucleus.
Prokaryotic and eukaryotic translation: Ribosome structure and assembly, tRNA, aminoacyl-tRNA synthetases, initiation, elongation and termination of polypeptides, fidelity of translation, inhibitors, posttranslational modifications
Prokaryotic and eukaryotic translation
Ribosome structure and assembly
Ribosomes are the cellular machinery for protein synthesis. Prokaryotic ribosomes are 70S, composed of 50S and 30S subunits, while eukaryotic ribosomes are 80S, composed of 60S and 40S subunits. Ribosome assembly involves ribosomal RNA (rRNA) and ribosomal proteins. In eukaryotes, processing of rRNA occurs in the nucleolus.
tRNA
Transfer RNA (tRNA) molecules are crucial for translating mRNA code into amino acids. Each tRNA carries a specific amino acid and has an anticodon that pairs with mRNA codons. The correct charging of tRNA with its respective amino acid is vital for accurate translation.
Aminoacyl-tRNA synthetases
Aminoacyl-tRNA synthetases are enzymes that charge tRNA with the correct amino acids, ensuring fidelity in protein synthesis. Each enzyme is specific to a particular amino acid and its corresponding tRNA, facilitating the first step in translating genetic information.
Initiation of translation
Initiation involves the assembly of the ribosomal subunits, mRNA, and the initiator tRNA. In prokaryotes, the Shine-Dalgarno sequence helps position the ribosome. Eukaryotic initiation requires the 5' cap and scanning for the start codon AUG.
Elongation of polypeptides
During elongation, amino acids are sequentially added to the growing polypeptide chain. The ribosome moves along the mRNA, with tRNA bringing new amino acids to the A site, facilitating peptide bond formation and translocation to the P site.
Termination of translation
Translation termination occurs when a stop codon (UAA, UAG, UGA) is reached. Release factors bind to the ribosome, promoting the release of the completed polypeptide and disassembly of the ribosomal complex.
Fidelity of translation
Fidelity in translation is critical for accurate protein synthesis. The correct matching between tRNA and mRNA codons ensures the proper sequence of amino acids. Aminoacyl-tRNA synthetases also play a key role in maintaining fidelity.
Inhibitors of translation
Translation can be inhibited by various compounds. In prokaryotes, antibiotics like tetracycline and chloramphenicol inhibit protein synthesis. Eukaryotic translation can be targeted by drugs like cycloheximide, affecting ribosomal function.
Posttranslational modifications
Posttranslational modifications include phosphorylation, glycosylation, and ubiquitination, which alter protein function, localization, and stability. These modifications are essential for the proper functioning of proteins within the cell.
Vectors: Cloning vectors (plasmids, cosmids, BACs, YACs), shuttle vectors, expression vectors
Vectors in Molecular Biology
Cloning Vectors
Cloning vectors are DNA molecules used to transport foreign genetic material into another cell. Common types include plasmids, cosmids, BACs, and YACs.
Plasmids
Plasmids are circular DNA molecules that replicate independently of chromosomal DNA. They are commonly used in gene cloning due to their ease of manipulation and the ability to carry inserts from 1,000 to 15,000 base pairs.
Cosmids
Cosmids are hybrid vectors that combine features of plasmids and bacteriophage lambda. They can carry larger DNA fragments than plasmids (up to 45,000 base pairs) and are useful for cloning larger genes.
BACs (Bacterial Artificial Chromosomes)
BACs are derived from F plasmids and can accommodate large insert sizes (up to 300,000 base pairs). They are especially valuable in genomic projects such as the Human Genome Project.
YACs (Yeast Artificial Chromosomes)
YACs can carry very large DNA inserts (up to 1 million base pairs). They facilitate the cloning of eukaryotic genes and are useful in studies of gene function and mapping.
Shuttle Vectors
Shuttle vectors are plasmids that can replicate in two different host species, usually a prokaryote and a eukaryote. This allows for the easy transfer of cloned genes into different systems for expression.
Expression Vectors
Expression vectors are designed to facilitate the expression of a gene of interest. They often contain a strong promoter for transcription and elements that ensure proper translation and post-translational modifications.
Enzymes used in DNA manipulating: Restriction endonucleases, ligases, polymerases, kinases, alkaline phosphatases, reverse transcriptase
Enzymes used in DNA manipulating
Restriction Endonucleases
Restriction endonucleases, also known as restriction enzymes, are proteins that cut DNA at specific sequences, known as restriction sites. They are crucial tools in molecular biology, allowing scientists to manipulate DNA sequences for cloning, gene insertion, and other genetic engineering applications. There are various types of restriction enzymes, which can produce different types of DNA ends, such as blunt or sticky ends.
Ligases
DNA ligases are enzymes that join two strands of DNA together by forming a phosphodiester bond. They are essential in DNA replication and repair, as well as in recombinant DNA technology when joining together fragments of DNA. Ligases are often used after restriction endonuclease digestion to construct recombinant plasmids.
Polymerases
DNA polymerases are enzymes that synthesize new strands of DNA by adding nucleotides to a pre-existing strand. They are critical for DNA replication and are also used in polymerase chain reaction (PCR) to amplify specific DNA sequences. Different polymerases have unique properties, such as proofreading ability and optimal temperature for activity.
Kinases
Kinases are enzymes that catalyze the transfer of phosphate groups from high-energy molecules, such as ATP, to specific substrates, usually proteins or nucleotides. In the context of DNA manipulation, kinases can be used to add phosphate groups to the ends of DNA fragments, aiding in the ligation process.
Alkaline Phosphatases
Alkaline phosphatases are enzymes that remove phosphate groups from nucleotides and proteins. In DNA manipulation, they are often used to dephosphorylate the ends of linearized plasmids to prevent self-ligation, which makes the cloning of foreign DNA more efficient.
Reverse Transcriptase
Reverse transcriptase is an enzyme that synthesizes complementary DNA (cDNA) from RNA templates. This enzyme is pivotal in molecular biology and genetic engineering, particularly in the creation of cDNA libraries or in reverse transcription PCR (RT-PCR), where RNA is converted to DNA for better analysis.
Genomic Library, PCR, Sequencing: Preparation and comparison of genomic and cDNA libraries, PCR and applications, DNA Sequencing, site directed mutagenesis, protein engineering concepts
Molecular Biology and Genetic Engineering
Genomic Library
A genomic library is a collection of DNA fragments that represent the complete genome of an organism. These fragments are inserted into vectors for cloning and storage, allowing researchers to access the genetic material for further analysis. Genomic libraries are useful for identifying genes, studying genetic variation, and exploring gene function.
cDNA Library
A cDNA library is a collection of complementary DNA (cDNA) synthesized from messenger RNA (mRNA) through the process of reverse transcription. cDNA libraries reflect gene expression profiles in specific tissues or developmental stages. They are instrumental in studying gene function, identifying expressed genes, and producing recombinant proteins.
PCR (Polymerase Chain Reaction)
PCR is a widely used technique for amplifying specific DNA sequences. It involves repeated cycles of denaturation, annealing, and extension, utilizing a DNA polymerase enzyme. Applications of PCR include cloning, gene expression analysis, genetic fingerprinting, and pathogen detection.
Sequencing
DNA sequencing is the process of determining the precise order of nucleotides in a DNA molecule. Techniques such as Sanger sequencing and next-generation sequencing (NGS) allow for efficient and accurate analysis of genomes. Sequencing is essential for understanding genetic variation, identifying mutations, and conducting genomic research.
Site-Directed Mutagenesis
Site-directed mutagenesis is a molecular biology technique used to introduce specific mutations into a DNA sequence. It enables the study of gene function and protein interactions. This technique is crucial in protein engineering, allowing for the design of proteins with enhanced properties or functions.
Protein Engineering
Protein engineering involves the design and construction of new proteins or the modification of existing proteins. By understanding protein structure and function, researchers can create proteins with desirable traits for applications in pharmaceuticals, agriculture, and industry. Techniques include recombinant DNA technology, directed evolution, and rational design.
Molecular Biology techniques: DNA isolation, blotting (Southern, Northern, Western), Electrophoresis of nucleic acids and proteins, Gene cloning, screening and characterization of cloned DNA, DNA Fingerprinting, RFLP, RAPD
Molecular Biology Techniques
DNA Isolation
DNA isolation involves extracting DNA from cells or tissues for further analysis. Common methods include phenol-chloroform extraction, silica gel-based methods, and column-based kits. The goal is to obtain pure and intact DNA for downstream applications.
Blotting Techniques
Blotting techniques are used to detect specific nucleic acids or proteins. Southern blotting is used for DNA, Northern blotting for RNA, and Western blotting for proteins. Each technique involves transferring molecules onto a membrane and probing with specific labeled sequences or antibodies.
Electrophoresis of Nucleic Acids and Proteins
Electrophoresis is a technique used to separate DNA, RNA, or proteins based on size and charge. Agarose gel electrophoresis is commonly used for nucleic acids, while SDS-PAGE is used for proteins. The results can be visualized using stains or markers.
Gene Cloning
Gene cloning involves the insertion of a DNA fragment into a vector to replicate it within a host organism. Methods include restriction enzyme digestion and ligation. This technique is fundamental for producing recombinant proteins and studying gene function.
Screening and Characterization of Cloned DNA
Screening involves identifying clones containing the desired insert. Techniques include colony PCR, restriction digestion analysis, and sequencing. Characterization ensures the cloned DNA is correct and functional.
DNA Fingerprinting
DNA fingerprinting is a method used to identify individuals based on unique patterns in their DNA. It involves analyzing specific regions of the genome, often using PCR and electrophoresis.
Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique that exploits variations in DNA sequences by digesting DNA with specific restriction enzymes. The resulting fragment patterns are compared among samples for genetic diversity studies or forensic analysis.
Random Amplified Polymorphic DNA (RAPD)
RAPD is a type of PCR-based technique that amplifies random segments of DNA. Variations in the amplification patterns can be used to assess genetic diversity and relationships among individuals.
