DNA Function and Gene Regulation- Bio Gr 12: Ch 7

Structure and Function of DNA

Discovery of DNA Structure

• The double helix structure of DNA was discovered through the work of scientists like Watson and Crick, but the functional implications of this structure were initially unclear.

• Early hints suggested that the sequence of nucleic acids (A, T, C, G) could determine the sequence of amino acids in proteins, laying the groundwork for understanding genetic coding.

• The significance of DNA's structure in relation to its function was not fully appreciated until later studies clarified these connections.

Historical Context: Archibald Garrod

• Archibald Garrod was a British physician who studied alkaptonuria, a genetic disorder that causes urine to turn black upon exposure to air.

• He proposed the concept of 'inborn errors of metabolism', suggesting that genetic mutations could lead to metabolic disorders due to faulty enzymes.

• Garrod's work laid the foundation for understanding the relationship between genes, enzymes, and phenotypes, encapsulated in the model: Genes → Enzyme → Phenotype.

Gene-Protein Relationship

Beadle and Tatum's Hypothesis

• George Beadle and Edward Tatum proposed the 'one gene-one enzyme hypothesis', asserting that each gene encodes a specific enzyme.

• Their experiments with the bread mold Neurospora crassa demonstrated that mutations could disrupt metabolic pathways, leading to the inability to synthesize certain compounds like arginine.

• This hypothesis evolved into the 'one gene-one polypeptide hypothesis', recognizing that not all proteins are enzymes, but each gene still codes for a specific polypeptide chain.

Central Dogma of Molecular Biology

• The central dogma describes the flow of genetic information: DNA → RNA → Protein.

• RNA serves as an intermediary, facilitating the transfer of information from the nucleus (where DNA resides) to the cytoplasm (where proteins are synthesized).

• The process of transcription converts DNA into mRNA, which is then translated into a protein sequence.

RNA: Structure and Function

Types of RNA and Their Roles

• RNA is crucial for protein synthesis and exists in several forms, including mRNA (messenger RNA), rRNA (ribosomal RNA), and tRNA (transfer RNA).

• mRNA carries the genetic information from DNA to the ribosome, where proteins are synthesized.

• tRNA transports specific amino acids to the ribosome, matching them to the corresponding codons on the mRNA during translation.

The Genetic Code

• The genetic code consists of 64 codons (triplets of nucleotides) that correspond to 20 amino acids, allowing for redundancy in coding.

• Start and stop codons signal the beginning and end of protein synthesis, respectively.

• The universality of the genetic code across species highlights its fundamental role in biology.

Transcription and Translation Processes

Transcription Process

• Transcription occurs in the nucleus and involves three main stages: initiation, elongation, and termination.

• RNA polymerase binds to the promoter region of DNA, unwinding the double helix and synthesizing a complementary RNA strand.

• The TATA box is a common feature of promoters, facilitating RNA polymerase binding and transcription initiation.

Translation Process

• Translation occurs in the cytoplasm, where ribosomes synthesize proteins based on the sequence of mRNA.

• The ribosome reads the mRNA codons, and tRNA molecules bring the appropriate amino acids to form a polypeptide chain.

• The process involves initiation, elongation, and termination, with specific sequences signaling the start and end of translation.

RNA Transcription and Processing

Overview of Transcription

• Transcription is the process where RNA is synthesized from a DNA template, resulting in the formation of pre-mRNA.

• The weak bond between RNA and DNA allows the RNA strand to detach, signaling the end of transcription.

• In prokaryotes, transcription occurs in the cytoplasm and does not require additional processing, while in eukaryotes, it occurs in the nucleus.

Post-Transcriptional Modifications

• Eukaryotic pre-mRNA undergoes modifications before exiting the nucleus to protect it from degradation.

• The 3' end of pre-mRNA receives a poly-A tail (50-250 adenines) added by poly-A polymerase, which aids in stability and protection from exonucleases.

• The 5' end is capped with a 7-methylguanylate cap, which protects the mRNA and serves as a recognition site for ribosomes during translation.

Introns and Exons

• Genes consist of coding regions (exons) and non-coding regions (introns); introns must be removed for functional protein synthesis.

• Prokaryotes lack introns, while eukaryotes have them, necessitating splicing to produce mature mRNA.

• Exons are joined together after intron removal, allowing for the correct amino acid sequence in proteins.

mRNA Splicing

• Splicing involves a complex of pre-mRNA and small nuclear ribonucleoproteins (snRNPs) forming a spliceosome.

• The spliceosome loops the intron out and joins the exons, ensuring a continuous coding sequence.

• Introns may provide benefits such as alternative splicing, which allows a single gene to produce multiple proteins, enhancing genetic efficiency.

Translation Process

Overview of Translation

• Translation is the process of synthesizing polypeptides based on the sequence of codons in mRNA.

• Each codon corresponds to a specific amino acid, and the ribosome plays a crucial role in this process.

• Transfer RNA (tRNA) molecules bring the appropriate amino acids to the ribosome, facilitating protein synthesis.

tRNA Structure and Function

• tRNA molecules are approximately 70-90 nucleotides long and have a cloverleaf structure with anticodons that pair with mRNA codons.

• The charging of tRNA with an amino acid is called aminoacylation, catalyzed by aminoacyl-tRNA synthetase.

• This process is energy-dependent and crucial for forming peptide bonds during translation.

The Ribosome and Its Role

• The ribosome consists of two subunits (large and small) and is responsible for catalyzing peptide bond formation between amino acids.

• It has three sites: A (aminoacyl), P (peptidyl), and E (exit), each playing a specific role in translation.

• The ribosome reads mRNA in the 5' to 3' direction, ensuring the correct sequence of amino acids in the polypeptide chain.

Stages of Translation

• Initiation begins with the small ribosomal subunit binding to mRNA and the initiator tRNA, followed by the large subunit's attachment.

• Elongation involves the addition of amino acids to the growing polypeptide chain, facilitated by peptidyl transferase.

• Termination occurs when a stop codon is reached, leading to the release of the completed polypeptide and disassembly of the ribosome.

tRNA Movement and Elongation

• The tRNA at the P site moves to the E site and is ejected, allowing the tRNA at the A site to move to the P site, making the A site available for a new charged tRNA.

• This cycle of tRNA movement continues, facilitating the elongation of the polypeptide chain during translation.

• The ribosome has three sites: A (aminoacyl), P (peptidyl), and E (exit), each playing a crucial role in the translation process.

Termination of Translation

• Elongation continues until the A site encounters a stop codon, which does not code for an amino acid but signals termination.

• The stop codon allows a release factor protein to bind to the mRNA, leading to the release of the polypeptide chain from the tRNA at the P site.

• Without a charged tRNA at the A site, the completed polypeptide detaches from the ribosome, which then disassembles from the mRNA strand.

Polysomes and Efficiency

• Multiple ribosomes can translate a single mRNA strand simultaneously, forming a structure known as a polysome.

• This arrangement significantly increases the speed and efficiency of protein synthesis, allowing for rapid production of proteins.

• In prokaryotes, translation occurs concurrently with transcription, enhancing efficiency compared to eukaryotes.

Comparative Analysis of Prokaryotic and Eukaryotic Processes

Key Differences in Transcription

• Prokaryotes have a single RNA polymerase for all transcription, while eukaryotes have three distinct RNA polymerases (I, II, III) for different types of RNA.

• Eukaryotic transcription occurs in the nucleus, followed by extensive processing, whereas prokaryotic transcription occurs in the cytoplasm without processing.

Translation in Prokaryotes vs. Eukaryotes

• In prokaryotes, translation can begin even before transcription is complete due to the lack of a nuclear membrane.

• Eukaryotic translation requires the mRNA to be fully processed and exported from the nucleus before it can be translated in the cytoplasm.

• The ribosomal structure and initiation mechanisms differ slightly between prokaryotes and eukaryotes, affecting the efficiency and regulation of protein synthesis.

Differences Between Prokaryotic and Eukaryotic Translation

Speed of Translation

• Prokaryotic translation is faster due to the availability of all components in the cytoplasm, allowing immediate access to ribosomes and mRNA.

• Eukaryotic translation is slower as it involves additional processing steps, including mRNA splicing and transport from the nucleus to the cytoplasm.

Polypeptide Processing

• Newly formed polypeptides often require extensive processing to become functional proteins, including clipping, folding, and cofactor addition.

• Enzymatic folding is crucial for the correct three-dimensional structure of proteins, which is essential for their function.

Gene Regulation

Overview of Gene Regulation

• Gene regulation ensures that not all genes are expressed at all times, allowing cells to respond to environmental changes efficiently.

• Housekeeping genes are continuously expressed to maintain basic cellular functions, while other genes are regulated based on need.

Prokaryotic Gene Regulation: Operons

• Prokaryotes utilize operons, which are clusters of genes controlled by a single promoter, to regulate gene expression.

• The lac operon is an example of an inducible operon, where the presence of lactose induces the transcription of genes needed for lactose metabolism.

• The trp operon is a repressible operon, where high levels of tryptophan inhibit the production of enzymes needed for its synthesis.

Eukaryotic Gene Control Mechanisms

Transcriptional Regulation

• Eukaryotic gene expression involves complex regulatory mechanisms, including transcription factors that bind to specific DNA sequences to enhance or inhibit transcription.

• Activators and repressors play critical roles in forming the transcription initiation complex with RNA polymerase.

Post-Transcriptional Regulation

• Alternative splicing allows for the production of multiple protein variants from a single gene by including or excluding specific exons.

• RNA interference mechanisms, such as miRNA and siRNA, can regulate gene expression by degrading mRNA or inhibiting translation.

Post-Translational Regulation

• Proteins often require post-translational modifications, such as cleavage or chemical modifications, to become active.

• The ubiquitin-proteasome pathway regulates protein degradation, determining protein lifespan and function.

Protein Degradation

Overview of Protein Degradation

• Proteins naturally degrade over time and usage, with lifespans ranging from minutes to decades.

• The degradation process is regulated by ubiquitin, a small protein that tags other proteins for degradation.

• A higher level of ubiquitin tagging correlates with shorter protein lifespan, while removal of ubiquitin can extend protein longevity.

• This process is crucial for maintaining cellular homeostasis and regulating protein levels in response to cellular needs.

• Proteins that evade degradation mechanisms can accumulate, potentially leading to cellular dysfunction.

Role of Ubiquitin in Protein Lifespan

• Ubiquitin acts as a signal for proteasomal degradation, marking proteins for destruction.

• The ubiquitin-proteasome pathway is a primary mechanism for protein turnover in eukaryotic cells.

• Ubiquitin can be added in chains, with polyubiquitination leading to targeted degradation.

• The process is reversible; deubiquitinating enzymes can remove ubiquitin, allowing proteins to persist longer in the cell.

• Dysregulation of ubiquitin pathways is implicated in various diseases, including cancer and neurodegenerative disorders.

Cancer Biology

Characteristics of Cancer Cells

• Cancer cells often lack regulatory mechanisms that control normal cell growth and division.

• Common mutations in cancer cells occur in promoter regions, coding regions, and genes responsible for telomere maintenance and growth stimulation (oncogenes).

• These mutations can lead to benign tumors, which are non-invasive, or malignant tumors, which can metastasize and require aggressive treatment.

• The distinction between benign and malignant cancers is crucial for determining treatment strategies, with benign tumors often treated surgically and malignant tumors requiring chemotherapy or radiation therapy.

• Understanding the genetic basis of cancer is essential for developing targeted therapies and personalized medicine.

Treatment Approaches for Cancer

• Treatment for benign tumors typically involves surgical removal, as they do not spread to other tissues.

• Malignant tumors often require a combination of therapies, including chemotherapy, radiation, and immunotherapy.

• Targeted therapies aim at specific genetic mutations within cancer cells, improving treatment efficacy and reducing side effects.

• Ongoing research into cancer genomics is leading to the development of novel therapeutic strategies, including gene editing and personalized medicine approaches.

• Early detection and intervention are critical for improving cancer prognosis and survival rates.

Mutations and Their Implications

Understanding Mutations

• Mutations are changes in the DNA sequence of an organism, which can arise from various factors such as exposure to mutagens, replication errors, or random changes.

• While many mutations are neutral due to the non-coding nature of most DNA, some can have significant effects on an organism's survival and fitness.

• Mutations are the only source of unique genetic variation, which is essential for evolution and adaptation in biological systems.

• The perception of mutations as harmful is often misleading; many contribute to genetic diversity and resilience in populations.

• Understanding mutation types and their consequences is crucial for fields such as genetics, evolutionary biology, and medicine.

Types of Mutations

• Small Scale Mutations: Involve changes in small groups of nucleic acids, including point mutations, which can be classified as missense, nonsense, silent, or frameshift mutations.

• Large Scale Mutations: Involve larger segments of DNA, including amplifications, deletions, translocations, inversions, and trinucleotide repeat expansions.

• Point Mutations: Can lead to significant changes in protein function, with missense mutations altering amino acid sequences, nonsense mutations creating premature stop codons, and silent mutations having no effect on the protein produced.

• Frameshift Mutations: Result from insertions or deletions that shift the reading frame, often leading to widespread changes in the resulting polypeptide chain.

• Amplifications and Deletions: Can lead to gene dosage effects, where the number of gene copies affects the phenotype, while deletions can result in loss of function.

Genome Components and Their Functions

Non-Coding Regions and Their Importance

• Non-coding regions make up a significant portion of the genome and include variable number tandem repeats (VNTRs) that contribute to genetic diversity.

• These regions play roles in buffering against replication errors and may influence gene expression and regulation.

• Telomeres and centromeres are examples of non-coding regions that protect chromosome integrity and facilitate proper segregation during cell division.

• Understanding the function of non-coding DNA is essential for a comprehensive view of genetics and its implications for evolution and disease.

• The presence of retrotransposons, such as LINEs and SINEs, adds complexity to genome organization and function, with potential roles in gene regulation and evolutionary processes.

Pseudogenes and Viral Contributions

• Pseudogenes are non-functional sequences that resemble functional genes but have accumulated mutations, providing insights into evolutionary history.

• Some pseudogenes may still play roles in gene regulation or exhibit neofunctionalization, adopting new functions over time.

• Viral DNA can integrate into host genomes, sometimes becoming heritable and contributing to genetic diversity, although often non-coding.

• The study of pseudogenes and viral contributions enhances our understanding of genome evolution and the mechanisms of genetic change.

• Comparative genomics allows researchers to explore the differences between genomes of various organisms, shedding light on functional implications and evolutionary relationships.

Non-Coding Regions and Their Roles

• Non-coding regions make up a significant portion of the genome, including variable number tandem repeats (VNTRs) that contribute to genetic diversity and stability during replication.

• Telomeres, repetitive sequences at the ends of chromosomes, protect against degradation and play a role in cellular aging.

• Centromeres, located at the site of sister chromatid binding, are essential for proper chromosome segregation during cell division.

• LINEs (Long Interspersed Nuclear Elements) and SINEs (Short Interspersed Nuclear Elements) are types of retrotransposons that can move within the genome, potentially influencing gene expression and evolution.

• The exact functions of LINEs and SINEs are not fully understood, but they are believed to contribute to genetic diversity and regulatory mechanisms.

Types of Genetic Mutations

Frameshift Mutations

• Frameshift mutations occur due to the insertion or deletion of one or more base pairs in the DNA sequence, leading to a shift in the reading frame of the genetic code.

• This shift alters the grouping of codons, potentially resulting in a completely different translation from the original sequence, which can lead to missense or nonsense mutations.

• Missense mutations result in the substitution of one amino acid for another in the resulting polypeptide, while nonsense mutations create a premature stop codon, truncating the protein.

• Frameshift mutations are typically damaging rather than benign, as they can disrupt the function of essential proteins.

• Example: Cystic fibrosis is often caused by a frameshift mutation in the CFTR gene, leading to dysfunctional chloride channels.

• The severity of the effects depends on the location of the mutation within the gene and the nature of the protein affected.

Large Scale Mutations

• Large scale mutations include amplifications, deletions, translocations, inversions, and trinucleotide repeat expansions, each having significant impacts on genomic structure and function.

• Amplifications: Genes can be duplicated and integrated into the genome, leading to increased expression and potential overactivity of the gene product. This can result in conditions like cancer, where oncogenes are often amplified.

• Deletions: The loss of segments of DNA can lead to the loss of gene function, which is typically harmful unless there are multiple copies of the gene present.

• Translocations: Involves the movement of entire genes or chromosomal segments to different locations, which can disrupt gene function or create fusion genes, as seen in certain leukemias.

• Inversions: A segment of DNA is flipped, potentially leading to non-functional proteins due to altered amino acid sequences.

• Trinucleotide repeat expansions: Repeated sequences can expand, leading to disorders like Huntington's disease, where the expanded repeats disrupt normal gene function.

Causes of Mutations

Spontaneous vs. Induced Mutations

• Spontaneous mutations arise from natural errors during DNA replication, which can occur due to the inherent inaccuracies of DNA polymerases.

• Induced mutations are caused by external factors, known as mutagens, which can be chemical agents or radiation.

• Chemical mutagens can mimic nucleotides, leading to incorrect base pairing, or can chemically modify nucleotides, resulting in misincorporation during replication.

• Radiation can cause direct DNA damage, such as strand breaks or fusion of DNA ends, leading to deletions or other mutations.

• Both spontaneous and induced mutations can result in small-scale changes (point mutations) or large-scale genomic alterations.

Viral Interactions with Host Genomes

Viral DNA Integration and Effects

• Viral infections can lead to the integration of viral DNA into the host genome, sometimes resulting in heritable changes that can affect future generations.

• This integrated viral DNA is often non-coding but can influence gene regulation and expression.

• Pseudogenes, which are non-functional sequences resembling functional genes, may arise from viral integration and can provide insights into evolutionary history.

• Example: Dolphins possess pseudogenes related to olfactory detection, indicating their terrestrial ancestry despite their aquatic lifestyle.

• The study of viral contributions to host genomes is crucial for understanding evolutionary processes and disease mechanisms.

Comparative Genomics and Viral Characteristics

Understanding Genomic Differences

• Comparative genomics involves analyzing the differences in genomes across various organisms to understand functional implications and evolutionary relationships.

• The sequence of DNA itself has limited value without context; how segments are utilized by the organism is critical for understanding biological functions.

• Viruses, while not classified as living organisms, exhibit characteristics of life, such as the ability to replicate and evolve.

• Viruses consist of a protein shell (capsid) that protects their nucleic acid, and some possess an envelope derived from the host cell membrane.

• High mutation rates in viruses can lead to rapid evolution and the emergence of new strains, complicating treatment and prevention strategies.

Discussion questions

1. How did Archibald Garrod's work on alkaptonuria contribute to our understanding of genetics and metabolism?

2. What is the significance of the 'one gene-one enzyme hypothesis' proposed by Beadle and Tatum?

3. Discuss the role of RNA in the process of protein synthesis.

4. How do post-transcriptional modifications affect gene expression in eukaryotic cells?

5. What are the implications of the Wobble Hypothesis for the genetic code and protein synthesis?

6. In what ways do prokaryotic and eukaryotic gene regulation differ?

Example answer

1. Garrod's study of alkaptonuria introduced the concept of 'inborn errors of metabolism,' suggesting that genetic mutations could lead to metabolic disorders. His hypothesis that genes are linked to enzyme function laid the groundwork for later discoveries about the relationship between genes, enzymes, and phenotypes.

2. The 'one gene-one enzyme hypothesis' was significant because it established a direct link between genes and the production of enzymes, demonstrating that each gene encodes a specific enzyme responsible for a particular metabolic pathway. This concept was foundational in molecular biology, leading to the understanding that genes also code for proteins, which are crucial for cellular functions.

3. RNA serves as a crucial intermediary in protein synthesis, facilitating the transfer of genetic information from DNA to ribosomes, where proteins are assembled. Messenger RNA (mRNA) carries the genetic code, while transfer RNA (tRNA) brings the appropriate amino acids, and ribosomal RNA (rRNA) forms the core of the ribosome, ensuring accurate translation of the mRNA sequence into a polypeptide chain.

4. Post-transcriptional modifications, such as capping, polyadenylation, and splicing, play a critical role in regulating gene expression by enhancing mRNA stability, facilitating nuclear export, and allowing for alternative splicing. These processes ensure that only properly processed mRNA is translated into proteins, thus influencing the diversity and functionality of proteins produced by eukaryotic cells.

5. The Wobble Hypothesis implies that the genetic code is more flexible than previously thought, allowing a single tRNA to recognize multiple codons due to variability in the third nucleotide position. This redundancy increases the efficiency of protein synthesis and provides a buffer against mutations, as changes in the third position often do not alter the resulting amino acid.

6. Prokaryotic gene regulation primarily relies on operons, which allow for coordinated control of gene expression through a single promoter, while eukaryotic regulation is more complex, involving multiple regulatory elements and mechanisms such as transcription factors, enhancers, and chromatin remodeling. This complexity in eukaryotes enables more nuanced control over gene expression in response to environmental and developmental cues.