DNA/RNA Metabolism Notes

Central Dogma of Molecular Biology: DNA → RNA → Protein

Flow of Genetic Information

• Genome: The entirety of genetic material in an organism, encompassing both coding and non-coding sequences vital for cellular function and regulation.

• Chromosomes: Organized DNA structures that are compacted and protected by histones, which provide structural support and play a role in gene regulation.

• Genes: Specific DNA sequences that encode proteins, comprising exons (coding regions) and introns (non-coding regions), which are transcribed into RNA.

General Scheme by Which Genes Control Cell Function

• Gene (DNA) -Transcription→ RNA formation -translation→ Protein Formation → Cell Structure OR Cell Enzymes → Cell Function

Cellular Protein Synthesis

• Activation of Signaling Pathways: Triggered by various stressors such as reactive oxygen species, mechanical stress, or hormonal changes, leading to the modification of protein synthesis.

• Transcriptional Activators: Molecules, often proteins, that bind to specific gene promoters to initiate the process of transcription by recruiting RNA polymerase.

• Transcription Process:

  • Transcription: The process where DNA is transcribed into messenger RNA (mRNA), which serves as a template for protein synthesis.

  • Translation: The mRNA is translated into a protein sequence, facilitated by transfer RNA (tRNA) that recognizes codons on the mRNA and brings the corresponding amino acids.

Redox-Mediated Signaling

• Redox signaling involves the regulation of cellular processes through the balance of reactive oxygen species (ROS) and antioxidants. This balance can influence various signaling pathways, affecting transcription factors and modifying protein activity. Redox states can alter the function of proteins, thereby impacting cellular responses to stress and influencing gene expression dynamics, particularly in processes like inflammation and cellular adaptation.

Nucleic Acids

• Types:

  • DNA: Deoxyribonucleic Acid - serves as the genetic blueprint for life; double-stranded and contains the four nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C).

  • RNA: Ribonucleic Acid - involved in the transmission of genetic information and protein synthesis; single-stranded and includes bases adenine (A), uracil (U), guanine (G), and cytosine (C).

• Components of Nucleotides:

  • Pentose Sugar: Deoxyribose in DNA and ribose in RNA, which are integral to nucleotide structure.

  • Nitrogenous Bases: Purines (Adenine, Guanine) with two rings and Pyrimidines (Thymine, Cytosine, Uracil) with one ring, critical for base pairing and genetic coding.

  • Phosphate Group: Linked to sugars and connects nucleotides, forming the backbone of DNA and RNA strands.

DNA Structure

• Double Helix:

  • Formed by two polynucleotide strands wrapped around each other, stabilized by hydrogen bonds between complementary bases.

  • Complementary bases pair: A with T (in DNA) and G with C (both DNA and RNA) following strict base-pairing rules.

  • Phosphodiester Bonds: Connect nucleotides together, resulting in a 5' to 3' orientation, essential for replication and transcription processes.

DNA Replication

• Occurs primarily during the S phase of the cell cycle, ensuring that each daughter cell receives an identical set of genetic material.

• Steps of DNA Replication:

  • Helicase resides in the internal points of a chromosome and hydrolyzes ATP to produce energy for separation of DNA strands

    • Unwinds the DNA and places Single Strand Binding Proteins to prevent reannealing

  • Strand separation creates a replication fork, allowing for formation of new DNA structures

  • Primase synthesizes an RNA primer, which serves as a template for the initiation of nucleotide replication

  • DNA Polymerase uses each strand as a template to synthesize new complementary strands

    • Primer strand attached to DNA template via complementary base binding

    • Complementary dNTP is attached to first existing base on DNA strand by hydrogen bonding

    • dNTP phosphorus binds to oxygen at 3’ end of primer

    • Successive dNTPs are attached in this manner

  • DNA Polymerase can only read template strands in a 3’ to 5’ direction, so the leading strand, which leads 3’ to 5’ can be read without interruption

  • The lagging strand has 5’ to 3’ structure, so the polymerase must work backwards to create the complementary strand

  • Lagging strand DNA is formed in segments called Okazaki Fragments, which are later joined together by the enzyme DNA ligase to create a continuous strand

  • Topoisomerase breaks the phosphodiester linkage of the DNA coil to reduce torsional stress, then rewinds once complete (keeps DNA coil from getting tangled as it unwinds)

Transcription Process (Making RNA from DNA)

• Key Factors:

  • RNA Synthesis: Facilitated by RNA polymerase, which binds to the promoter region (e.g., TATA box), facilitating the initiation of transcription.

  • Pre-mRNA Processing: Involves the splicing out of introns, addition of a 5' cap, and a poly-A tail to produce a mature mRNA ready for translation.

  • Regulation Factors: Enhancers, insulators, and a variety of other transcription factors influence the transcription rate, determining the level of gene expression.

• Steps of Transcription

  • RNA Polymerase 2 binds to the promoter sequence, typically a series of T and A nucleotides (called a TATA box)

  • The RNA polymerase temporarily “unwinds” the DNA double helix

  • The polymerase “reads” the DNA strand and adds complementary RNA molecules to the DNA template

  • “Activated” RNA molecules react with the growing end of the RNA strand and are added (3’ end)

    • RNA Polymerase 2 adds two additional phosphates to the phosphate backbone of the nucleotides being attached

    • Provides enough energy to form 5’→3’ phosphodiester linkage between existing nucleotide 3- exposed end and new nucleotide

  • Transcription ends when the RNA Polymerase reaches a chain terminating sequence, releasing both the polymerase and the RNA strand

    • Terminating sequence is typically a series of G’s and C’s, creating a hairpin structure that stops transcription

  • Pre-mRNA (non-functional RNA) is spliced into mature mRNA structure

    • This process is directed by another type of RNA called small nuclear RNA (snRNA)

    • snRNAs combine with nuclear proteins to form small nuclear ribonucleoproteins (snRNP)

    • 5 snRNPs combine to form the spliceosome, which directs mRNA splicing

    • A 5’ cap (methylated guanosine) and a 3’ poly-A tail are added to prevent destruction by RNAses

Euakryotic Transcriptional Regulation

• Transcription factor IID binds to TATA box to prepare the promoter region for RNA polymerase 2

• Transcription factors IIA and IIB join with IID and the DNA strand, which then allows for adherence of RNA polymerase 2

• Nearby and upstream enhancers regulate the transcribing speed and function of RNA polymerase 2, via binding of other transcriptional regulators to this region

• Insulators prevent action of enhancers from neighboring regions from affecting local transcription

Novel, Small RNA Molecules

• Small Nuclear RNA (snRNA): assists with RNA splicing in the nucleus

• Small Nucleolar RNA (snoRNA): required for rRNA processing in the nucleolus

• MicroRNA (miRNA): RNA sequences that block translation, called gene silencing

  • Bind to 3’ untranslated region of mRNA

  • Made from duplexed RNA (folded, helix RNA)

    • Drosha (nuclear) and Dicer (cytosol) are ribonucleases that hydrolyze the duplex RNA into miRNA

  • Small Interfering RNA (siRNA): gene silencers from duplex RNA derived from laboratory or viral RNA

    • Hydrolyzed by Dicer

Gene Expression Control

• Gene expression can be induced (upregulated) by environmental signals or suppressed (downregulated) as part of regulatory mechanisms.

• Regulation of several different stages of the central “central dogma” allow for gene expression control

  • Transcription

  • RNA processing into mRNA, tRNA, rRNA, or small RNA sequences

  • RNA transport to cytosol

  • RNA degradation - constantly occurs, but can be altered

  • Translation

  • Proteolysis - constantly occurs as well, but proteins are more stable than RNA

  • Post-translational modification and protein targeting

    • Many proteins need to be carboxylated or glycosylated in order to be properly utilized in their biological roles

How does exercise alter the stages of gene expression?

• Exercise significantly influences various stages of gene expression:

  • Large increases in transcriptional regulatory proteins are observed post-exercise

  • Mechano Growth Factor (MGF), an isoform of IGF-1, is produced almost exclusively after exercise

    • Results from alternative splicing of IGF-1 gene RNA transcript; promotes myogenesis at all stages

  • Transport is enhanced by production of SKAR

    • A nuclear protein that facilitates mRNA transport to the cytosolic S6K1, to enhance translational efficiency

  • RNA degradation - exercise alters miRNA production, thus effecting RNA degradation rate

  • Translation - increased production of eukaryotic initiation factors for enhancing translational efficiency

  • Proteolysis - exercise speeds up proteolysis

    • Protease activation, autophagy, ubiquitin-proteasome pathway

  • Post-translation modification and protein targeting

    • Markedly enhanced protein phosphorylation and protein acetylation occurs after resistance and endurance exercise

    • PGC-1a nuclear targeting to the nucleus is enhanced after exercise

Summary of Muscle Hypertrophy Mechanisms

• mTOR: Central regulator that promotes mRNA translation during periods of muscle growth, responding to nutrient and hormonal signals.

• Satellite Cells: These progenitor cells proliferate in response to muscle injury induced by exercise, playing a crucial role in muscle regeneration and repair to sustain muscle mass.

Epigenetic Changes

• DNA Methylation: Inhibits gene expression through the addition of methyl groups to cytosine residues in the DNA, impacting gene accessibility and transcriptional activity.

• Histone Modification: Acetylation of histones removes positive charges, thereby loosening the interaction with DNA, increasing accessibility for transcription factors and enhancing gene expression.