DNA Replication

Semiconservative Replication

DNA replication is the process by which a cell duplicates its DNA, ensuring that each daughter cell receives an exact copy. The process is described as semiconservative because each new DNA molecule consists of one original (parental) strand and one newly synthesized strand.

• Template Function: Each parental strand serves as a template for the synthesis of a new complementary strand.

• Result: After two rounds of replication, each DNA molecule contains a mix of old and new strands.

• Significance: This mechanism preserves genetic information across generations.

Steps in Bacterial DNA Replication

Replication in bacteria begins at a specific location called the origin of replication and proceeds biodirectly around cromosome

Initiation: Replication starts at the origin, forming two replication forks that move in opposite directions.

• Termination: Replication ends when the forks meet at the termination site, resulting in two identical DNA molecules.

Enzymes and Proteins Involved

• Helicase: Unwinds and unzips the DNA double helix, creating a replication fork.

• Single-Stranded Binding Proteins (SSBPs): Stabilize the unwound DNA strands to prevent reannealing.

• Primase: Synthesizes short RNA primers needed to initiate DNA synthesis.

• DNA Polymerase III: Adds nucleotides to the 3’ end of the primer in a 5’ to 3’ direction and proofreads newly synthesized DNA.

• DNA Ligase: Joins Okazaki fragments on the lagging strand.

Leading and Lagging Strand Synthesis

DNA polymerase can only add nucleotides to the 3’ end, resulting in continuous synthesis on one strand (leading) and discontinuous synthesis on the other (lagging).

• Leading Strand: Synthesized continuously toward the replication fork.

• Lagging Strand: Synthesized discontinuously away from the fork in short segments called Okazaki fragments.

Energy for DNA Synthesis

Nucleotide addition is powered by the hydrolysis of high-energy phosphate bonds in nucleoside triphosphates.

• Pyrophosphate Release: The removal of pyrophosphate provides energy for the formation of phosphodiester bonds.

Gene Expression: Transcription and Translation

Overview of Gene Expression

Gene expression involves two main processes: transcription (DNA to mRNA) and translation (mRNA to protein). This flow of genetic information is central to cellular function.

• mRNA (messenger RNA): Carries genetic information from DNA to ribosomes.

• tRNA (transfer RNA): Brings amino acids to the ribosome during translation.

• rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

Transcription: DNA to mRNA

Transcription is the synthesis of RNA from a DNA template, catalyzed by RNA polymerase.

• Initiation: RNA polymerase binds to the promoter region of DNA, aided by sigma factors in prokaryotes.

• Elongation: RNA polymerase synthesizes RNA in the 5’ to 3’ direction, adding ribonucleotides complementary to the DNA template (A:U, C:G).

• Termination: RNA polymerase recognizes termination signals, releasing the completed RNA transcript.

Translation: mRNA to Protein

Translation is the process by which ribosomes synthesize proteins using the sequence of codons in mRNA as a template.

• Initiation: Ribosomal subunits assemble on the mRNA, and the initiator tRNA binds to the start codon (AUG).

• Elongation: tRNAs bring amino acids to the ribosome, where peptide bonds are formed between amino acids in the growing polypeptide chain.

• Termination: When a stop codon (UAA, UAG, UGA) is reached, the completed polypeptide is released.

Genetic Code

The genetic code is a set of rules by which the nucleotide sequence of mRNA is translated into the amino acid sequence of proteins. Each set of three nucleotides (codon) specifies a particular amino acid.

• Start Codon: AUG (methionine in eukaryotes, N-formylmethionine in prokaryotes)

• Stop Codons: UAA, UAG, UGA

• Degeneracy: Multiple codons can code for the same amino acid.

Prokaryotic vs. Eukaryotic Gene Expression

• Prokaryotes: Transcription and translation can occur simultaneously in the cytoplasm; mRNA is often polycistronic (codes for multiple proteins).

• Eukaryotes: Transcription occurs in the nucleus, translation in the cytoplasm; mRNA is typically monocistronic and undergoes processing (splicing, capping, polyadenylation).

• Introns and Exons: Eukaryotic genes contain introns (non-coding) and exons (coding); introns are removed during mRNA processing.

Regulation of Gene Expression: Operons

Operon Structure and Function

An operon is a cluster of genes under the control of a single promoter and operator, allowing coordinated regulation of gene expression in prokaryotes.

• Structural Genes: Code for enzymes and polypeptides.

• Operator: DNA region where a repressor protein can bind to block transcription.

• Promoter: DNA region where RNA polymerase binds to initiate transcription.

• Regulatory Gene: Codes for a repressor protein, often located away from the operon.

Inducible Operons: The Lac Operon

The lac operon is an example of an inducible operon, which is usually off but can be turned on in the presence of an inducer (lactose).

• Normal State: Repressor binds to the operator, blocking transcription.

• Induction: Lactose (inducer) binds to the repressor, inactivating it and allowing transcription of genes needed to metabolize lactose.

Repressible Operons: The trp Operon

The trp operon is an example of a repressible operon, which is usually on but can be turned off when the end product (tryptophan) is abundant.

• Normal State: Repressor is inactive, and transcription proceeds, producing enzymes for tryptophan synthesis.

• Repression: Excess tryptophan acts as a corepressor, activating the repressor, which then binds the operator and blocks transcription.

Horizontal Gene Transfer in Bacteria

Mechanisms of Horizontal Gene Transfer

Horizontal gene transfer allows bacteria to acquire new genetic traits from other organisms, contributing to genetic diversity and adaptation.

• Transformation: Uptake of naked DNA from the environment by competent cells.

• Transduction: Transfer of bacterial DNA by bacteriophages (viruses that infect bacteria).

• Conjugation: Direct transfer of DNA between bacterial cells via a pilus.

Transformation

Transformation involves the uptake of free DNA fragments or plasmids from the environment. Discovered by Griffith in Streptococcus pneumoniae, this process requires cells to be competent (able to bind and import DNA).

• Artificial Competence: Can be induced in the lab using CaCl2 and heat shock or electroporation.

• Application: Widely used in recombinant DNA technology.

Transduction

Transduction is the transfer of bacterial genes by bacteriophages. It can occur naturally in many bacterial species and requires the donor and recipient to be of the same species.

• Generalized Transduction: Any bacterial gene can be transferred.

• Specialized Transduction: Only specific genes near the phage integration site are transferred.

Conjugation

Conjugation is the transfer of DNA through direct cell-to-cell contact, primarily in Gram-negative bacteria. It requires a pilus to connect donor (F+) and recipient (F-) cells.

• F Plasmid: The fertility plasmid enables the donor to form a pilus and transfer DNA.

• Result: The recipient becomes F+ and can participate in further conjugation.

High Frequency Recombination (Hfr) Cells

Hfr cells have the F factor integrated into their chromosome, allowing them to transfer chromosomal genes at high frequency during conjugation. This process is reversible, and only genes adjacent to the F factor are transferred.

Plasmids

Plasmids are small, circular, double-stranded DNA molecules that replicate independently of the bacterial chromosome. They often carry genes for antibiotic resistance, virulence factors, or metabolic pathways, but are not essential for basic survival.

• Transfer: Plasmids can be transferred by transformation, transduction, or conjugation.