Comprehensive Study Guide: DNA, RNA, Replication, Repair, Transcription, and Translation

DNA and RNA: Structure and Function

Distinguishing Features of DNA and RNA

DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are nucleic acids that store and transmit genetic information. They differ in several structural and functional aspects:

• Sugar Component: DNA contains deoxyribose; RNA contains ribose.

• Nitrogenous Bases: DNA uses thymine (T); RNA uses uracil (U) instead.

• Strandedness: DNA is typically double-stranded and forms a stable double helix; RNA is usually single-stranded and less stable.

• Stability: DNA is more chemically stable due to the absence of a 2'-hydroxyl group on its sugar.

Example: mRNA is a type of RNA that carries genetic information from DNA to the ribosome for protein synthesis.

DNA Electrophoresis and Physical Properties

Agarose Gel Electrophoresis

Agarose gel electrophoresis is a technique used to separate DNA fragments based on size and conformation.

• Migration Pattern: Supercoiled DNA migrates faster than linear or nicked circular DNA due to its compact shape.

• Influencing Factors: Gel concentration, voltage, buffer composition, and DNA conformation affect migration.

Example: Plasmid DNA can appear as multiple bands corresponding to different conformations (supercoiled, linear, nicked).

DNA Replication: Mechanism and Regulation

Meselson-Stahl Experiment and Semi-Conservative Replication

The Meselson-Stahl experiment demonstrated that DNA replication is semi-conservative, meaning each daughter DNA molecule contains one parental and one newly synthesized strand.

• Method: Used isotopic labeling of nitrogen to distinguish old and new DNA strands.

• Conclusion: After one round of replication, DNA molecules had intermediate density, supporting the semi-conservative model.

DNA Polymerases and Nucleotide Incorporation

DNA polymerases are enzymes that synthesize new DNA strands by adding nucleotides to a primer strand in the 5' to 3' direction.

• Primer Requirement: DNA polymerases require a short RNA primer to initiate synthesis.

• Directionality: DNA is always synthesized in the 5' to 3' direction.

• Energy Source: The energy for polymerization comes from the hydrolysis of dNTPs (deoxynucleoside triphosphates).

Equation:

$(dNMP)_n + dNTP \rightarrow (dNMP)_{n+1} + PP_i$

Origin of Replication and Bidirectionality

Replication begins at specific sequences called origins of replication (ORI). In eukaryotes, the origin recognition complex (ORC) binds to the ORI to initiate replication.

• Bidirectional Replication: Replication proceeds in both directions from the origin, forming two replication forks.

• Activation: Helicase unwinds the DNA, and single-stranded binding proteins stabilize the unwound strands.

Replication Fork and Okazaki Fragments

The replication fork is the Y-shaped region where DNA is actively unwound and replicated.

• Leading Strand: Synthesized continuously in the direction of fork movement.

• Lagging Strand: Synthesized discontinuously as Okazaki fragments, later joined by DNA ligase.

• Asymmetry: The antiparallel nature of DNA causes this difference in synthesis.

Replication Complex Components

The replication complex includes multiple proteins:

• DNA polymerase

• Helicase

• Primase

• Clamp loader and sliding clamp

• Single-stranded binding proteins (SSBs)

• RNase H (removes RNA primers)

Cell Cycle and Replication Timing

DNA replication occurs during the S phase of the cell cycle. The duration varies by organism and cell type.

DNA Repair Mechanisms

Types of DNA Repair

Cells employ several repair mechanisms to maintain genome integrity:

• Base Excision Repair (BER): Repairs small, non-helix-distorting base lesions.

• Nucleotide Excision Repair (NER): Removes bulky, helix-distorting lesions (e.g., thymine dimers).

• Mismatch Repair (MMR): Corrects errors introduced during DNA replication.

Steps in NER:

1. Damage recognition

2. Excision of damaged DNA segment

3. DNA synthesis to fill the gap

4. Ligation

DNA Glycosylases and AP Endonucleases

DNA glycosylases remove damaged bases, creating abasic (AP) sites. AP endonucleases cleave the DNA backbone at these sites to facilitate repair.

Homologous and Non-Homologous End Joining

Double-strand breaks are repaired by:

• Homologous Recombination (HR): Uses a homologous template for accurate repair.

• Non-Homologous End Joining (NHEJ): Directly ligates broken ends, often resulting in small insertions or deletions.

Transcription and Its Regulation

Transcription Process

Transcription is the synthesis of RNA from a DNA template, involving three main steps:

• Initiation: RNA polymerase binds to the promoter region.

• Elongation: RNA polymerase synthesizes the RNA strand.

• Termination: RNA polymerase releases the completed RNA transcript.

Promoters and Transcription Factors

Promoters are DNA sequences that define where transcription begins. Transcription factors and RNA polymerase recognize these sequences to initiate transcription.

• Core Promoter: Includes the TATA box and initiator elements.

• Enhancers/Silencers: Regulatory elements that increase or decrease transcription efficiency.

Epigenetic Regulation

Epigenetic modifications, such as DNA methylation and histone modification, regulate gene expression without altering the DNA sequence.

• DNA Methylation: Addition of methyl groups to cytosine bases, often silencing gene expression.

• Histone Modification: Acetylation, methylation, and phosphorylation of histone proteins affect chromatin structure and gene accessibility.

RNA Processing and Translation

RNA Processing in Eukaryotes

Primary RNA transcripts (pre-mRNA) undergo several modifications before becoming mature mRNA:

• 5' Capping: Addition of a 7-methylguanosine cap for stability and ribosome recognition.

• Splicing: Removal of introns and joining of exons by the spliceosome.

• 3' Polyadenylation: Addition of a poly(A) tail for stability and export from the nucleus.

Translation: The Genetic Code and Ribosomes

Translation is the process by which ribosomes synthesize proteins using mRNA as a template.

• Codons: Triplets of nucleotides in mRNA that specify amino acids.

• tRNA: Transfer RNA molecules bring amino acids to the ribosome and match codons via their anticodon.

• Ribosome Sites: A (aminoacyl), P (peptidyl), and E (exit) sites coordinate tRNA binding and peptide bond formation.

Equation:

$\text{Protein synthesis:} \quad \text{mRNA} + \text{tRNA} + \text{ribosome} \rightarrow \text{polypeptide chain}$

Regulation of Translation

Translation is regulated at multiple levels, including mRNA structure, initiation factors, and regulatory RNAs (miRNA, siRNA).

• miRNA/siRNA: Small RNAs that can bind to mRNA and inhibit translation or promote degradation.

• Polysomes: Multiple ribosomes translating a single mRNA simultaneously, increasing efficiency.

Summary Table: DNA vs. RNA

Feature	DNA	RNA
Sugar	Deoxyribose	Ribose
Bases	A, T, C, G	A, U, C, G
Strandedness	Double-stranded	Single-stranded (usually)
Stability	More stable	Less stable
Function	Genetic information storage	Information transfer, catalysis, regulation

Additional info:

• Some questions in the original file reference specific textbook chapters and sections (e.g., "12 Molecular Cell Biology - Ch. 5 part 2 DNA Repair").

• For exam preparation, students should be able to explain mechanisms, compare processes, and understand the roles of key enzymes and regulatory elements in DNA replication, repair, transcription, and translation.