Exam 3 Study Guide - Module 5 PDF

**[5.2 DNA Replication]** I. **Basics of DNA Replication** - - - - - - - - - - - - - - - - - - - II. **DNA Replication in Prokaryotes** - - - Reported evidence of bacterial origins of replication in 1963. - Demonstrated formation...

**[5.2 DNA Replication]** I. **Basics of DNA Replication** - - - - - - - - - - - - - - - - - - - II. **DNA Replication in Prokaryotes** - - - Reported evidence of bacterial origins of replication in 1963. - Demonstrated formation of a replication bubble as replication initiates in bacteria. - E. coli has 4.6 million base pairs and replicates in \~42 minutes. - Replication starts from a single origin, proceeding bidirectionally at \~1000 nucleotides per second. - **DNA Polymerase (DNA pol)**: - Adds nucleotides complementary to the template strand. - Requires energy from nucleotides with three phosphates. - **Types of DNA Polymerases**: - DNA pol III: Main enzyme for DNA synthesis. - DNA pol I and II: Primarily for DNA repair. - **Helicase**: Unwinds DNA at the replication fork. - **Primase**: Synthesizes a short RNA primer to provide a free 3\'-OH group. - **Single-Strand Binding Proteins**: Stabilize unwound DNA to prevent re-formation of double helix. - **Topoisomerase**: Relieves supercoiling ahead of the replication fork by breaking and reforming the DNA backbone. - **DNA Ligase**: Joins Okazaki fragments by sealing nicks in the sugar-phosphate backbone. - Forms when helicase separates the DNA strands. - Two replication forks are created at the origin, extending bi-directionally. - - - - RNA primers are replaced with DNA by DNA pol I. - Exonuclease activity of DNA pol I removes RNA primers. - DNA ligase seals nicks between newly synthesized DNA and previously synthesized DNA. - III. **Replication initiation in bacteria** - **Origin of Replication (oriC)**: - Bacterial DNA replication begins at a specific location called the origin of replication, typically referred to as **oriC** in *E. coli*. - **Binding of Initiator Proteins**: - Specialized initiator proteins, such as **DnaA** in *E. coli*, bind to the oriC region. This binding is essential for the initiation process. - **DNA Unwinding**: - The binding of DnaA proteins causes localized unwinding of the DNA, creating single-stranded regions. - These single-stranded areas allow other proteins to bind and facilitate further unwinding. - **Helicase Loading**: - After initial unwinding, the enzyme **helicase** (DnaB) is recruited to the single-stranded DNA. - Helicase unwinds the DNA by breaking hydrogen bonds between the nitrogenous bases, further opening up the double helix. - **Formation of the Primase Complex**: - The enzyme **primase** (DnaG) synthesizes short RNA primers complementary to the single-stranded DNA. This provides the necessary 3\'-OH group for DNA polymerase to begin synthesis. - **DNA Polymerase Recruitment**: - The main DNA polymerase (DNA pol III) is then recruited to the replication fork, where it starts adding nucleotides to the growing DNA strand using the RNA primer as a starting point. - **Formation of the Replication Fork**: - As replication proceeds, two replication forks are formed, allowing bidirectional replication to occur from the origin. - **Regulatory Mechanisms**: - The initiation process is tightly regulated to ensure that replication occurs only once per cell cycle. Factors such as DnaA concentration and the presence of specific sequences at the oriC are critical in this regulation. In summary, replication initiation in bacteria involves the binding of initiator proteins to the origin, unwinding of the DNA, loading of helicase, and the synthesis of RNA primers, leading to the recruitment of DNA polymerase and the formation of replication forks. IV. **Prokaryotic DNA Replication Enzymes** - **DNA Unwinding**: - DNA unwinds at the origin of replication. - **Helicase Action**: - Helicase opens the DNA, forming replication forks that extend bidirectionally. - **Single-Strand Binding Proteins**: - These proteins coat the DNA around the replication fork to prevent re-winding. - **Topoisomerase Function**: - Topoisomerase binds ahead of the replication fork to prevent supercoiling. - **Primase Activity**: - Primase synthesizes RNA primers that are complementary to the DNA strand. - **DNA Polymerase III**: - DNA polymerase III begins adding nucleotides to the 3\'-OH end of the RNA primer. - **Strand Elongation**: - Elongation continues for both the lagging and leading strands. - **Removal of RNA Primers**: - RNA primers are removed by the exonuclease activity of DNA polymerase I. - **Filling Gaps**: - Gaps are filled by DNA polymerase I through the addition of deoxynucleoside triphosphates (dNTPs). - **Sealing the Fragments**: - DNA ligase seals the gaps between DNA fragments by forming phosphodiester bonds. **Enzymes Involved in Prokaryotic DNA Replication** **Enzyme** **Function** ------------------------------------------- ------------------------------------------------------------------------ **Helicase (DnaB)** Unwinds the DNA at the replication fork. **Single-Strand Binding Proteins (SSBs)** Stabilize single-stranded DNA and prevent rewinding. **Topoisomerase** Relieves supercoiling ahead of the replication fork. **Primase (DnaG)** Synthesizes RNA primers complementary to the DNA strand. **DNA Polymerase III** Main enzyme for DNA synthesis, adds nucleotides to the growing strand. **DNA Polymerase I** Removes RNA primers and fills gaps with DNA. **DNA Ligase** Seals nicks between DNA fragments by forming phosphodiester bonds. This table provides a clear overview of the key enzymes and their respective roles in prokaryotic DNA replication. V. **DNA replication, important players and steps** - **Helicase (DnaB)**: - **Function**: Unwinds the double-stranded DNA at the replication fork by breaking the hydrogen bonds between base pairs, allowing the strands to separate. - **Single-Strand Binding Proteins (SSBs)**: - **Function**: Bind to and stabilize the single-stranded DNA during replication, preventing the strands from re-annealing or forming secondary structures. - **Topoisomerase**: - **Function**: Relieves supercoiling tension ahead of the replication fork by cutting the DNA, allowing it to unwind, and then resealing it. This prevents tangling and ensures smooth progression of replication. - **Primase (DnaG)**: - **Function**: Synthesizes short RNA primers that are complementary to the DNA template. These primers provide a free 3\'-OH group necessary for DNA polymerase to begin adding nucleotides. - **DNA Polymerase III**: - **Function**: The primary enzyme responsible for DNA synthesis. It adds nucleotides to the 3\'-OH end of the RNA primer, extending the DNA strand in the 5\' to 3\' direction. - **DNA Polymerase I**: - **Function**: Responsible for removing RNA primers from the lagging strand and replacing them with DNA nucleotides. It also plays a role in DNA repair. - **DNA Ligase**: - **Function**: Seals the nicks between adjacent DNA fragments (Okazaki fragments on the lagging strand) by forming phosphodiester bonds, thus creating a continuous DNA strand. - **Sliding Clamp (e.g., PCNA in eukaryotes, but in prokaryotes, it's also a type of clamp)**: - **Function**: Holds DNA polymerase in place on the DNA template, ensuring that it remains attached during strand elongation for efficient nucleotide addition. - **Exonuclease (activity of DNA Polymerase I)**: - **Function**: Removes the RNA primers during DNA replication and replaces them with DNA nucleotides. **Summary** These key players work in concert to ensure accurate and efficient DNA replication, each fulfilling a specific role in unwinding the DNA, synthesizing new strands, and ensuring the integrity of the genetic material throughout the process. VI. **Simultaneous synthesis of leading and lagging strands** - **Replication Fork Formation**: - DNA replication occurs at the replication fork, where the double helix is unwound by helicase, creating two single-stranded templates. - **Leading Strand Synthesis**: - The leading strand is synthesized continuously in the 5\' to 3\' direction towards the replication fork. - DNA polymerase III adds nucleotides to the 3\'-OH end of the RNA primer, extending the new DNA strand as the fork progresses. - **Lagging Strand Synthesis**: - The lagging strand is synthesized discontinuously away from the replication fork in short segments known as **Okazaki fragments**. - Each Okazaki fragment starts with an RNA primer synthesized by primase, allowing DNA polymerase III to add nucleotides in the 5\' to 3\' direction. - **Simultaneous Action**: - Both strands are synthesized simultaneously due to the coordinated action of multiple DNA polymerase complexes, each functioning at the replication fork: 1. **Leading strand**: One polymerase complex continuously synthesizes the leading strand. 2. **Lagging strand**: Multiple polymerase complexes synthesize the lagging strand in short bursts (Okazaki fragments). - **Role of the Sliding Clamp**: - A sliding clamp protein holds DNA polymerase III in place on the DNA template, ensuring efficient and continuous nucleotide addition for both strands. - **Processing of Okazaki Fragments**: - Once an Okazaki fragment is completed, DNA polymerase I removes the RNA primer and fills in the gap with DNA nucleotides. - DNA ligase then seals the nicks between adjacent Okazaki fragments, forming a continuous lagging strand. **Summary** This mechanism allows for the efficient and coordinated replication of both the leading and lagging strands, ensuring that the entire DNA molecule is accurately copied during cell division. The simultaneous synthesis is crucial for maintaining the speed and fidelity of DNA replication. **Holoenzyme** - **Definition**: A holoenzyme is a complete and active enzyme complex that includes both the enzyme\'s protein component (apoenzyme) and its required co-factors or coenzymes. These components are essential for the enzyme to function properly. - **Components**: - **Apoenzyme**: The protein part of the enzyme that provides the specific catalytic activity. - **Cofactors**: Non-protein molecules that assist in enzyme activity. They can be metal ions (e.g., Mg²⁺) or organic molecules (coenzymes like NAD⁺ or FAD). - **Function in DNA Replication**: - In the context of DNA replication, DNA polymerase III in prokaryotes is an example of a holoenzyme. It consists of multiple subunits that together allow it to effectively synthesize DNA. The holoenzyme structure enables simultaneous synthesis of both the leading and lagging strands by coordinating the activity of the polymerase, clamp, and other associated proteins. **Importance** - Holoenzymes are crucial for the activity of many enzymes, as the co-factors or coenzymes often provide the necessary chemical groups for catalysis or structural stability, enabling the enzyme to perform its biological function effectively. VII. **DNA Replication in Eukaryotes** - **Complexity and Size**: - Eukaryotic genomes are larger and more complex than prokaryotic genomes. - The human genome has approximately 3 billion base pairs per haploid set, totaling 6 billion during replication in the S phase. - **Origins of Replication**: - Eukaryotic chromosomes have multiple origins of replication, with humans having up to 100,000 origins. - In yeast, sequences called **Autonomously Replicating Sequences (ARS)** serve as origins of replication, analogous to those in *E. coli*. - **DNA Polymerases**: - Eukaryotes possess more DNA polymerases than prokaryotes; 14 have been identified, with five playing major roles during replication: 1. **Pol α** 2. **Pol β** 3. **Pol γ** 4. **Pol δ** 5. **Pol ε** - **Pre-Replication Complex**: - Before replication, DNA is bound to histones, forming nucleosomes. - Chemical modifications of chromatin may occur to allow access for replication enzymes. - A pre-replication complex is formed at the origin with various initiator proteins. - **Helicase Activity**: - Helicase unwinds the DNA helix using energy from ATP hydrolysis, forming replication forks. - **Topoisomerase Function**: - Topoisomerases resolve supercoiling ahead of the replication fork, preventing tangling of DNA. - **Primer Synthesis**: - Primase, in conjunction with low-processivity enzyme Pol α, synthesizes RNA primers. - Pol α then extends the primer with a short stretch of DNA before being replaced by the main DNA polymerases. - **Leading and Lagging Strand Synthesis**: - **Leading Strand**: Mainly synthesized by DNA Pol ε, though recent evidence suggests Pol δ may also play a role. - **Lagging Strand**: Synthesized discontinuously in Okazaki fragments. - **Sliding Clamp**: - **PCNA (Proliferating Cell Nuclear Antigen)** holds DNA polymerases in place during replication, enhancing processivity. - **Primer Removal and Replacement**: - **Replication Protein A (RPA)** and nucleases **FEN1** and **Dna2** are involved in removing RNA primers and replacing them with DNA. - **Joining Okazaki Fragments**: - After RNA primers are replaced with DNA, DNA ligase seals the remaining gaps by forming phosphodiester bonds, completing the replication of the lagging strand. **Summary Table of Key Players** **Component** **Function** ------------------- --------------------------------------------------- **Histones** Package DNA into nucleosomes **Helicase** Unwinds DNA strands at replication fork **Topoisomerase** Relieves supercoiling tension ahead of the fork **Primase** Synthesizes RNA primers **Pol α** Extends RNA primers and initiates DNA synthesis **Pol ε/Pol δ** Main enzymes for leading strand synthesis **PCNA** Sliding clamp that holds DNA polymerase in place **RPA** Stabilizes single-stranded DNA **FEN1 & Dna2** Remove RNA primers and facilitate DNA replacement **DNA Ligase** Seals gaps between DNA fragments This summary outlines the key processes and components involved in eukaryotic DNA replication, highlighting its complexity compared to prokaryotic replication. VIII. **DNA proofreading and finishing replication** - **Proofreading Mechanism**: - **3\' to 5\' Exonuclease Activity**: DNA polymerases possess a proofreading function through their exonuclease activity. If an incorrect nucleotide is incorporated during DNA synthesis, the polymerase can detect the mismatch. - **Correction Process**: 1. The polymerase pauses and reverses its direction, removing the incorrectly added nucleotide from the 3\' end of the growing DNA strand. 2. After excising the wrong nucleotide, DNA polymerase can then add the correct nucleotide in the 5\' to 3\' direction. - **High Fidelity**: - DNA polymerases have a high fidelity rate, meaning they make very few errors during DNA replication. The combination of correct base pairing and proofreading reduces the error rate to approximately 1 mistake per billion nucleotides added. - **Role of Accessory Proteins**: - Accessory proteins, such as **Replication Protein A (RPA)** and **PCNA**, help maintain the stability and integrity of the DNA replication process, further reducing the likelihood of errors. **Finishing DNA Replication** 1. **Completing the Lagging Strand**: - After the synthesis of Okazaki fragments on the lagging strand, the RNA primers are removed by nucleases (like FEN1 and Dna2). - DNA polymerase I fills in the gaps left by the removed primers with DNA nucleotides. 2. **Joining Fragments**: - DNA ligase seals the nicks between adjacent Okazaki fragments, creating a continuous DNA strand. 3. **Telomere Replication**: - At the ends of linear chromosomes, special structures called **telomeres** are replicated by an enzyme called **telomerase**, which adds repetitive sequences to protect the chromosome ends from degradation. 4. **Final Checks**: - Additional repair mechanisms, such as mismatch repair pathways, can further correct any remaining errors after replication is complete. **Summary** - **DNA polymerase** prevents replication errors primarily through its **proofreading capability**, which allows it to detect and correct mistakes in real-time. The combination of this proofreading activity, high fidelity, and the action of various accessory proteins ensures that DNA replication is accurate and efficient. After replication, mechanisms are in place to finalize the process and maintain genomic integrity. IX. **Telomere Replication** A diagram of a dna sequence Description automatically generated - **Linear Chromosomes**: - Eukaryotic chromosomes are linear, unlike prokaryotic circular chromosomes. - DNA polymerase can only synthesize DNA in the 5\' to 3\' direction. - **Lagging Strand Challenge**: - On the lagging strand, DNA is synthesized in short stretches (Okazaki fragments), each initiated by separate RNA primers. - At the ends of linear chromosomes, there is no template for a primer, leading to unpaired ends. - **Telomeres**: - The ends of linear chromosomes are known as **telomeres**, consisting of repetitive sequences that do not code for any genes. - In humans, the sequence **TTAGGG** is repeated 100 to 1000 times. - Telomeres protect the genes from deletion during cell division, preventing chromosome shortening. - **Telomerase**: - **Telomerase** is an enzyme that maintains the length of telomeres. - It contains a catalytic component and an RNA template. - Telomerase attaches to the end of the chromosome and adds complementary DNA bases to the 3\' end using its RNA template. - Once the lagging strand template is sufficiently elongated, DNA polymerase can extend the strand by adding nucleotides complementary to the DNA. - **Activity of Telomerase**: - Telomerase is typically active in **germ cells** and **adult stem cells**, which require continuous cell division. - It is generally **inactive in adult somatic cells**, leading to progressive shortening of telomeres with each cell division. - **Nobel Prize Recognition**: - Elizabeth Blackburn was awarded the Nobel Prize in Medicine and Physiology in 2009 for her discovery of telomerase and its role in chromosome maintenance. **Importance** - The discovery of telomeres and telomerase has significant implications for understanding aging, cancer, and cellular senescence, as the loss of telomeric sequences can lead to genomic instability. X. **Telomerase and Aging** - **Telomere Shortening and Aging**: - Most somatic cells do not produce telomerase, leading to the progressive shortening of telomeres with each cell division. - This shortening is associated with aging and age-related conditions. - **Increasing Human Lifespan**: - Advances in modern medicine and healthier lifestyles have contributed to increased human lifespans. - There is a growing demand for younger appearance and better quality of life in older age. - **Research on Telomerase in Mice**: - In 2010, studies showed that telomerase can reverse some age-related conditions in mice, particularly in telomerase-deficient mice. - These mice exhibited: 1. Tissue atrophy 2. Stem cell depletion 3. Organ system failure 4. Impaired responses to tissue injury - Reactivation of telomerase in these mice led to: 5. Extension of telomeres 6. Reduction in DNA damage 7. Reversal of neurodegeneration 8. Improved function of organs such as testes, spleen, and intestines - Suggests potential for telomere reactivation in treating age-related diseases in humans. - **Telomeres and Cancer**: - Cancer is characterized by uncontrolled division of abnormal cells, accumulation of mutations, and the ability to metastasize. - Cancerous cells are often found to have significantly shortened telomeres. - Telomerase activity is observed in cancer cells, but it becomes active only after telomeres have been shortened. - Inhibiting telomerase activity in cancer cells could potentially halt further cell division during cancer therapy. **Implications** - **Regenerative Medicine**: Reactivating telomerase may offer therapeutic avenues for age-related conditions, improving health and longevity. - **Cancer Therapy**: Targeting telomerase in cancer cells could be a strategic approach to limit tumor growth and metastasis. **[5.3 Point Mutations and Proofreading]** I. **Mutations are Rare and Random** - **Definition of Mutation**: - A mutation is a heritable change in the DNA sequence of an organism. - The resulting organism with the mutation is termed a **mutant**. - **Wild Type vs. Mutant**: - The **wild type** refers to the most commonly observed phenotype in nature. - Mutants may exhibit recognizable phenotypic changes compared to the wild type. - **Process of Mutation Impact**: - Mutations are transcribed into mRNA through **transcription**. - This may lead to an altered amino acid sequence during **translation**, potentially resulting in a changed protein function. - **Role of Proteins**: - Proteins perform the majority of cellular functions; thus, changes in their amino acid sequences can lead to altered phenotypes for the cell and organism. - **Nature of Mutations**: - Mutations are typically random and often deleterious, impairing the function of genes or gene products. - They generate inherited genetic diversity, contributing to **evolutionary change**. - **Types of Mutations**: - **Gene mutations** can substitute, add, or delete one or more DNA base pairs. - These localized mutations are referred to as **point mutations**. - **Point Mutations**: - Occur at specific, identifiable positions in a gene. - Consequences of point mutations vary based on: 1. Type of sequence change (substitution, insertion, deletion) 2. Location within the gene (coding region, regulatory region, etc.) - **Mutation Rates**: - Mutation rates can differ among organisms and among different genes within a single species. **Importance** - Understanding mutations is crucial for studying genetic diversity, evolution, and the molecular basis of diseases. II. **Point Mutations: Base-Pair Substitution Mutations** - **Base-Pair Substitutions**: - The most common type of nucleotide mutation, involving the replacement of one nucleotide base pair with another. - **Types of Base-Pair Substitutions**: - **Transitions**: 1. A purine is replaced by another purine (e.g., adenine (A) replaced by guanine (G)). 2. A pyrimidine is replaced by another pyrimidine (e.g., cytosine (C) replaced by thymine (T)). - **Transversions**: 3. A purine is replaced by a pyrimidine or vice versa (e.g., cytosine (C) replaced by adenine (A) or thymine (T)). - **Further Categorization of Base-Pair Substitutions**: - **Silent Mutation**: 4. Does not alter the amino acid sequence of the protein due to redundancy in the genetic code. - **Missense Mutation**: 5. Results in the substitution of one amino acid for another in the protein sequence, potentially affecting protein function. - **Nonsense Mutation**: 6. Converts an amino acid codon into a stop codon, leading to premature termination of protein synthesis. **Importance** - Understanding the types of nucleotide mutations and their consequences is essential for studying genetic disorders, protein function, and evolutionary biology. III. **Tautomeric Shift-Induced Mismatches** - **Inherent Mutation Rate**: - DNA replication has a maximum accuracy of about **10\^-4 mutations per base pair** due to tautomeric shift-induced mismatches. - **Tautomers**: - Tautomers are isomers that differ in the positions of atoms and the bonds between them. - Each of the four bases in DNA can exist in at least two tautomeric forms: 1. **Adenine (A) and Cytosine (C)**: 1. Can exist as **amino** or **imino** forms (cyclic amidines). 2. **Guanine (G), Thymine (T), and Uracil (U)**: 2. Can exist as **keto** or **enol** forms (cyclic amides).  - **Stability of Tautomers**: - The **amino** and **keto** forms are more stable and predominate in cellular conditions, while the **imino** and **enol** forms are rarer. - **Mispairing**: - The imino and enol forms can mispair with the wrong base: 3. Example: If cytosine shifts to its rare imino form, it may pair with adenine instead of guanine. - **Correction Mechanism**: - Mismatches caused by tautomeric shifts can be corrected by the **3\'-5\' exonuclease activity** of DNA polymerase, allowing the correct base to be incorporated. **Importance** - Understanding tautomeric shifts and their impact on mutation rates is crucial for studying DNA replication fidelity and the mechanisms of genetic variation. IV. **Types of Base-Pair Substitution Mutations** A diagram of a dna sequence Description automatically generated - **Types of Point Mutations**: - **Silent Mutation**: - A point mutation that does not change the amino acid incorporated into the polypeptide. - Due to the degeneracy of the genetic code, it has no effect on the protein's structure. - **Missense Mutation**: - Results in the incorporation of a different amino acid into the polypeptide. - The impact depends on: 1. **Chemical differences** between the new amino acid and the wild-type amino acid. 2. **Location** within the protein (e.g., changes in the enzyme's active site can have significant effects). - Many missense mutations lead to proteins that retain some functionality. - Some missense mutations may exhibit effects only under specific environmental conditions and are termed **conditional mutations**. - Occasionally, missense mutations can confer a **selective advantage** under certain conditions. - **Nonsense Mutation**: - Converts a codon encoding an amino acid into a **stop codon** (nonsense codon). - Results in the synthesis of truncated proteins that are typically non-functional. **Importance** - Understanding the effects of different types of point mutations is essential for elucidating the relationships between genotype and phenotype, as well as the mechanisms of evolution and disease. V. **Point Mutations: Frameshift Mutations** - **Types of Mutations**: - **Insertion**: - Addition of one or more nucleotide bases to the DNA sequence. - **Deletion**: - Removal of one or more nucleotide bases from the DNA sequence. - **Effects of Insertions and Deletions**: - **Codons and Triplet Structure**: - Codons consist of triplets of nucleotides. - Insertions or deletions of **three nucleotides** may result in the addition or removal of one or more amino acids without significantly affecting protein functionality. - **Frameshift Mutations**: - Caused by insertions or deletions of nucleotides that are **not multiples of three**. - Result in a **shift in the reading frame** of the mRNA. - Alters the downstream codons, potentially changing every amino acid after the mutation. - May introduce a **premature stop codon** before the full coding sequence is translated. - Proteins resulting from frameshift mutations are nearly always **nonfunctional**. **Importance** - Understanding the consequences of insertions, deletions, and frameshift mutations is crucial for studying genetic disorders, protein functionality, and the mechanisms of evolution. VI. **Point Mutations: Regulatory Mutations** - Regulatory mutations alter the amount of protein produced by a gene without changing the amino acid sequence. - They affect regions such as promoters, introns, and untranslated regions (UTRs) of mRNA. - Types of regulatory mutations: promoter mutations, splicing mutations, cryptic splice sites, and polyadenylation mutations. - **Types of Regulatory Mutations**: - **Promoter Mutations**: - Alter consensus sequence nucleotides, impacting transcription initiation. - Example: Human β-globin gene has mutations that moderately reduce transcript and protein levels. - Result in decreased β-globin gene expression. - **Splicing Mutations**: - Reduce or eliminate normal splicing of pre-mRNA. - Key sequences: GT at the 5\' splice site and AG at the 3\' end of exons. - Mutations in these sequences can lead to splicing errors. - Example: Mutations in intron 1 of the β-globin gene disrupt normal splicing processes. - **Cryptic Splice Sites**: - Arise from base-pair substitutions that create new splice sites competing with original ones. - Example: A mutation in intron 1 of the β-globin gene can create a cryptic splice site, leading to abnormal splicing in 90% of events. - Alters the normal splicing pattern, potentially impacting protein function. - **Polyadenylation Mutations**: - Caused by mutations in the polyadenylation signal sequence (5\' AAUAAA 3\'). - Example: A mutation from 5\' AATAAA 3\' to 5\' AATAAG 3\' in the α-globin gene disrupts proper mRNA processing. - Leads to abnormal mRNA and significantly reduced protein function. **Importance** - Regulatory mutations are critical for understanding gene expression regulation, potential disease mechanisms, and therapeutic interventions. VII. **DNA Proofreading** - **Importance of Accuracy**: - DNA replication is crucial for maintaining genetic integrity. - Errors during replication can lead to mutations, which may result in serious consequences such as cancer. - **Proofreading by DNA Polymerases**: - Most DNA polymerases possess a proofreading function. - As nucleotides are added, the enzyme checks for proper base pairing with the template strand. - If an incorrect base is detected, the polymerase can correct the error. - **Mechanism of Proofreading**: - The polymerase shifts the newly synthesized strand to its 3\' → 5\' exonuclease site. - This site cleaves the phosphodiester bond, removing the incorrectly incorporated nucleotide. - After excision, the polymerase can then insert the correct nucleotide, ensuring fidelity in the DNA sequence. - **Consequences of Errors**: - If proofreading fails, errors may remain uncorrected, potentially leading to mutations. - Mutations can disrupt normal cellular functions and may contribute to the development of diseases, including cancer. - **Repair Mechanisms**: - In addition to proofreading, cells have multiple DNA repair pathways to correct mismatches and damage. - Defective repair mechanisms or mutations in repair enzymes can exacerbate the risk of accumulating harmful mutations. **Summary** Through a combination of proofreading and various repair mechanisms, cells maintain the high fidelity of DNA replication, safeguarding against mutations and the associated risks of diseases like cancer. **[5.4 Causes of Mutations and Direct Repair of DNA Damage]** I. **Causes of Mutations** **Spontaneous Mutations**: - **Cause**: Errors that occur naturally during DNA replication. - **Error Rate**: DNA polymerase has an error rate of about one incorrect base per billion base pairs replicated. - **Impact**: Most errors are corrected by proofreading mechanisms, but some can lead to mutations if not repaired. **Induced Mutations**: - **Cause**: Result from exposure to mutagens, which can be chemical agents or physical agents (like radiation). - **Mutation Rate**: Exposure to mutagens can increase mutation rates by more than 1000-fold. II. **Mutations induced by chemical or ionizing radiation** **What is a Mutagen?** A **mutagen** is any agent that increases the frequency of mutations in an organism\'s DNA. Mutagens can be classified into two main categories: **chemical mutagens** and **physical mutagens** (including ionizing radiation). They can alter the structure of DNA, leading to errors during DNA replication or causing damage that can result in permanent changes in the genetic material. Mutations can be induced by both chemical agents and ionizing radiation, each of which alters DNA in different ways. Here's a breakdown of how these mutagens work: **1. Chemical Mutagens** **Types of Chemical Mutagens:** - **Base Analogs**: These are chemicals that resemble normal DNA bases and can be incorporated into DNA during replication. For example, 5-bromouracil (a thymine analog) can pair with adenine instead of guanine, leading to mutations. - **Alkylating Agents**: These compounds add alkyl groups to DNA bases, altering their pairing properties. Common examples include: - **Mustard Gas**: Used in warfare, it can cause significant DNA damage. - **Ethyl Methanesulfonate (EMS)**: Frequently used in laboratory settings to induce mutations. - **Intercalating Agents**: These agents insert themselves between DNA base pairs, causing frameshift mutations. Examples include: - **Acridine Dyes**: Used in genetic research. - **Ethidium Bromide**: Commonly used for staining DNA in gel electrophoresis. - **Deaminating Agents**: These chemicals remove amino groups from bases, changing their pairing properties. For instance: - **Nitrous Acid**: Can convert adenine to hypoxanthine, which pairs with cytosine instead of thymine. **2. Ionizing Radiation** **Types of Ionizing Radiation:** - **X-rays and Gamma Rays**: These high-energy radiation forms can cause breaks in the DNA backbone, leading to mutations. They can also generate free radicals, which can damage DNA further. - **Cosmic Rays**: High-energy particles from space can similarly break DNA strands or cause other types of damage. **Effects of Mutations Induced by These Agents** - **Point Mutations**: Changes in a single base pair can occur due to mispairing or base substitution. For example, an alkylating agent might cause a guanine to pair incorrectly during DNA replication. - **Frameshift Mutations**: Insertions or deletions of bases, especially when caused by intercalating agents, can shift the reading frame of codons, leading to completely altered protein sequences. - **Chromosomal Aberrations**: Ionizing radiation can lead to larger-scale changes in chromosome structure, including deletions, duplications, or translocations, which can have severe consequences for the cell. **Summary** Both chemical and ionizing radiation-induced mutations can significantly impact an organism\'s genetic makeup, leading to various diseases, including cancer. Understanding these mechanisms is crucial for developing strategies to prevent and treat mutagen-induced conditions. III. **Causes of Mutations: Chemical Mutagens - Nucleoside Analogs** Chemical mutagens can induce mutations in DNA through various mechanisms, primarily by acting as nucleoside analogs or by modifying existing nucleotide bases. Here's a detailed look at these processes: **1. Nucleoside Analogs** Nucleoside analogs are structurally similar to normal nucleotide bases, allowing them to be incorporated into DNA during replication. However, they often follow different base-pairing rules, leading to mutations: - **2-Aminopurine (2AP)**: This analog resembles adenine and can pair with cytosine (C) instead of thymine (T). Over several rounds of replication, this substitution converts an AT base pair into a GC base pair. - **5-Bromouracil (5BU)**: Similar to thymine, this analog can pair with guanine (G) rather than adenine (A). Consequently, an initial AT base pair can be transformed into a GC base pair after a few rounds of DNA replication. **2. Modifying Agents** Certain chemicals modify existing nucleotide bases, altering their base-pairing properties: - **Nitrous Acid**: This agent deaminates cytosine (C), converting it into uracil (U). In this modified form, uracil pairs with adenine (A) instead of guanine (G). As a result, this leads to a transition mutation where a CG base pair becomes a TA base pair. Additionally, nitrous acid can deaminate adenine (A) to hypoxanthine, which then pairs with cytosine (C) instead of thymine (T), resulting in a TA base pair converting to a CG base pair. **Summary** Chemical mutagens can significantly impact DNA integrity by introducing point mutations through mechanisms that either replace normal bases with analogs or modify existing bases to change their pairing properties. These mutations can have profound effects on gene expression and protein function, contributing to various diseases, including cancer. Understanding these mechanisms is essential for developing strategies to mitigate mutagenic effects and improve DNA repair processes. IV. **Causes of Mutations: Chemical Mutagens - Intercalating Agents** Intercalating agents are a distinct class of chemical mutagens that interact with DNA in a unique way. Here\'s an overview of how they function and their implications: **Mechanism of Action** - **Intercalation**: Intercalating agents insert themselves between the stacked nitrogenous bases of the DNA double helix. This insertion distorts the DNA structure, altering the spacing between the nucleotide base pairs. - **Impact on Replication**: During DNA replication, this distortion can lead to errors: - **Insertions**: DNA polymerase may inadvertently add extra nucleotides, resulting in insertions. - **Deletions**: Conversely, the polymerase might skip over several bases, leading to deletions. **Consequences** - **Frameshift Mutations**: Both insertions and deletions can cause frameshift mutations, where the reading frame of the gene is altered. This can result in completely different amino acid sequences downstream of the mutation, often leading to nonfunctional proteins. **Examples of Intercalating Agents** - **Polycyclic Aromatic Hydrocarbons (PAHs)**: These are combustion byproducts and are known to be potent intercalating agents that can contribute to cancer development by inducing mutations. - **Ethidium Bromide and Acridine Orange**: Commonly used in laboratories to visualize DNA in gel electrophoresis, these compounds are also recognized as potential mutagens due to their ability to intercalate into DNA. **Summary** Intercalating agents pose a significant risk for inducing mutations through their unique ability to distort DNA structure, leading to replication errors. Their mutagenic potential highlights the importance of handling these substances carefully in laboratory and environmental contexts, as well as understanding their role in cancer biology. V. **Causes of Mutations: Radiation** Exposure to radiation can indeed lead to mutations in DNA through different mechanisms depending on whether the radiation is ionizing or nonionizing. Here's a breakdown of how each type induces mutations: **Ionizing Radiation** - **Types**: Includes X-rays and gamma rays. - **Mechanism**: - **DNA Breaks**: Ionizing radiation can cause single-stranded and double-stranded breaks in the DNA backbone by generating hydroxyl radicals. - **Base Modification**: Similar to chemical mutagens, ionizing radiation can lead to the deamination of bases, such as converting cytosine to uracil, which can lead to base-pairing errors during replication. - **Effects**: The nonspecific damage caused by ionizing radiation can kill cells or lead to significant mutations if the damage is not repaired. This property is utilized in sterilizing medical devices and food. **Nonionizing Radiation** - **Types**: Primarily includes ultraviolet (UV) light. - **Mechanism**: - **Photoproduct Formation**: UV light causes the formation of aberrant structures in DNA called photoproducts. The two main types are: - **Thymine Dimers**: Covalent linkage between adjacent thymine bases. This can stall DNA replication and transcription, leading to mutations if not repaired. - **6-4 Photoproducts**: A bond forms between carbon 6 of one thymine and carbon 4 of another. This structure can also disrupt normal DNA replication. - **Error-Prone Repair**: When replication stalls due to photoproducts, specialized DNA polymerases may fill in the gaps through a process called translesion synthesis. These polymerases lack proofreading ability, making them more prone to errors, which can result in frameshift or point mutations. **Summary** Both ionizing and nonionizing radiation can induce mutations, but through different mechanisms. Ionizing radiation causes direct damage and breaks in DNA, while nonionizing radiation primarily leads to structural changes that disrupt replication. Understanding these mechanisms is crucial for assessing the risks associated with radiation exposure and developing strategies for DNA repair and mutation prevention. VI. **The Ames Test I** To assess whether a substance is a mutagen, the **Ames test** is commonly used. This test mimics how potential mutagens affect living organisms, specifically focusing on how they might induce mutations that could lead to cancer. Here\'s a breakdown of the process: **Ames Test Overview** 1. **Bacterial Strain**: The test employs *Salmonella typhimurium* strains that are histidine auxotrophs (His-negative). These bacteria cannot synthesize histidine and therefore require it from their environment to grow. 2. **Liver Extract**: To simulate mammalian metabolism, rat liver enzymes are used. Many chemicals require metabolic activation to become mutagenic, and this step is crucial for the test. 3. **Control Setup**: In a control experiment, *Salmonella* without any added mutagen will show a low number of colonies, as they cannot grow without histidine. 4. **Test Substance**: When a potential mutagen is added to the culture with the *Salmonella*, and the bacteria are allowed to grow, an increase in colonies indicates that mutations have occurred, allowing the bacteria to synthesize histidine again (this is termed \"reversion\"). 5. **Reversion Rate**: The effectiveness of the mutagen is quantified by calculating the ratio of revertants (colonies formed after exposure to the mutagen) to the control. This ratio helps assess the mutagen\'s strength. 6. **Different Mutant Strains**: The test can be refined further using different *Salmonella* strains that are sensitive to specific types of mutations (point mutations vs. frameshift mutations). This allows researchers to determine the specific mechanism by which a mutagen acts. **DNA Repair Mechanisms** When DNA damage occurs, such as from UV light (causing thymine dimers), two primary repair mechanisms can help fix the damage: 1. **Nucleotide Excision Repair (NER)**: - This pathway removes damaged DNA segments and replaces them with the correct nucleotides. - Enzymes recognize the distortion caused by the damage, excise the affected strand, and synthesize a new segment using the undamaged strand as a template. 2. **Photoreactivation**: - This mechanism directly reverses UV-induced thymine dimers. - An enzyme called photolyase binds to the dimer and uses light energy (typically blue light) to break the bonds between the thymines, restoring the original bases. **Human Implications** - **Xeroderma Pigmentosa**: A genetic disorder caused by mutations in genes essential for NER. Individuals with this condition are extremely sensitive to UV light and have a high risk of developing skin cancers due to their inability to repair UV-induced DNA damage effectively. **Higher-Energy Radiation** For higher-energy radiation (like X-rays): - **Damage**: It can cause single-stranded and double-stranded breaks in DNA. Double-strand breaks are particularly severe because they can prevent the DNA from being used as a template for repair. - **Specialized Repair Systems**: Cells possess sophisticated repair mechanisms (e.g., homologous recombination and non-homologous end joining) to address these types of damage, ensuring the integrity of the genome is maintained. **Conclusion** The Ames test serves as a valuable tool in identifying potential mutagens, while understanding DNA repair mechanisms is crucial for recognizing how cells respond to DNA damage. The interplay between mutagens and repair processes highlights the importance of maintaining genomic stability in living organisms. VII. **The Ames Test II** The **Ames test**, developed by Bruce Ames in the 1970s, is a key method for screening the carcinogenic potential of chemical compounds using bacteria. Here\'s a detailed overview of the process and its significance: **Overview of the Ames Test** 1. **Test Organism**: The test utilizes a specific strain of *Salmonella typhimurium* that is a histidine auxotroph, meaning it cannot synthesize histidine due to a mutation in a gene essential for histidine production. 2. **Procedure**: - **Exposure**: The *Salmonella* bacteria are exposed to the test compound, potentially a chemical that is suspected of being mutagenic. - **Plating**: After exposure, the bacteria are plated on a medium that lacks histidine. This setup ensures that only those bacteria that have reverted to a histidine-synthesizing form (mutants) can grow. - **Counting Mutants**: After incubation, the number of colonies that grow (indicating successful reversion) is counted. This number is compared to a control group where no mutagen was added. 3. **Rat Liver Extract**: Since many chemicals are not mutagenic in their original form but become mutagenic after metabolic activation (as they would in the liver), rat liver extract is often included in the test. This simulates the metabolic processing of the compound, allowing for a more accurate assessment of its mutagenic potential. 4. **Interpreting Results**: - An increased number of revertants (mutants that regain the ability to synthesize histidine) in the presence of the compound compared to the control indicates that the substance may be mutagenic. - The degree of increase in revertants can provide insights into how potent the mutagen is. **Importance and Applications** - **Rapid Screening**: The Ames test is valued for its rapid and inexpensive methodology, allowing researchers to quickly assess the mutagenic potential of new compounds. - **Initial Indicator of Carcinogenic Risk**: Although not definitive for human carcinogenicity, an elevated mutation rate suggests a higher risk for cancer, prompting further investigation. - **Further Testing**: Compounds identified as mutagenic in the Ames test are usually subjected to additional tests, including animal models (like mice and rats), to evaluate their potential carcinogenic properties more thoroughly. **Conclusion** The Ames test is a foundational tool in toxicology and pharmacology, helping to identify potentially harmful substances before they are used in products or released into the environment. By leveraging the principles of microbial genetics, it provides a critical first step in assessing chemical safety. VIII. **Direct Repair of DNA Damage: Mismatch Repair** Mismatch repair is a critical process that corrects errors that escape the proofreading ability of DNA polymerases during DNA replication. Here's a detailed overview of how this repair mechanism works, particularly in *E. coli* and eukaryotic cells: **Mismatch Repair Mechanism** 1. **Error Detection**: - After DNA replication, enzymes recognize mismatched base pairs (incorrectly added nucleotides) that have not been corrected. - In *E. coli*, this involves a system known as methyl-directed mismatch repair. 2. **Hemimethylation**: - In *E. coli*, the parental DNA strand is methylated while the newly synthesized daughter strand is not. This hemimethylated state helps the mismatch repair system identify which strand is the template and which is newly synthesized. - The methylation of the parental strand occurs several minutes after replication, providing a window for mismatch repair. 3. **Repair Proteins**: - **MutS**: This protein recognizes and binds to the mismatched base pair. - **MutL**: This protein interacts with MutS to form a complex that facilitates the next steps in the repair process. - **MutH**: This enzyme specifically recognizes the hemimethylated state of the DNA and makes a cut in the non-methylated (newly synthesized) strand at the site of the mismatch. 4. **Excision**: - An exonuclease removes a segment of the DNA strand, starting from the cut made by MutH and extending to the mismatch. This segment includes the incorrect nucleotide. 5. **Replacement**: - DNA polymerase III fills in the gap left by the excised segment using the methylated parental strand as a template. - Finally, DNA ligase seals the remaining nick in the sugar-phosphate backbone. **Eukaryotic Mismatch Repair** - The mismatch repair mechanism in eukaryotes is less well understood than in prokaryotes, but some key points include: - Eukaryotic cells also utilize a mismatch repair system, although the specific proteins and mechanisms involved can vary among different organisms. - It is believed that recognition of nicks or other signs of incomplete replication on the new strand is important for directing the repair. - Some replication proteins may continue to associate with the daughter strand for a short time after replication, which may aid in recognizing mismatches. **Importance of Mismatch Repair** - **Preventing Mutations**: Mismatch repair is vital for maintaining genomic integrity. Failure to correct mismatches can lead to permanent mutations, which may contribute to diseases, including cancer. - **Evolutionary Implications**: This repair mechanism contributes to the fidelity of DNA replication, which is crucial for the evolution and stability of species over time. By efficiently correcting replication errors, mismatch repair systems play a key role in the overall health and stability of an organism\'s genome. IX. **Direct Repair of DNA Damage: Repair of Thymine Dimers** Thymine dimers, formed when ultraviolet (UV) light induces covalent bonding between adjacent thymine bases, can disrupt DNA structure and function. To address this, organisms have evolved several repair mechanisms, primarily nucleotide excision repair and photoreactivation.  **Nucleotide Excision Repair (NER)** 1. **Detection of Damage**: - An enzyme complex scans the DNA for distortions, such as those caused by thymine dimers. - Upon detecting a dimer, the complex recognizes the structural change in the DNA helix. 2. **Cutting the DNA**: - The enzyme complex makes cuts in the sugar-phosphate backbone several bases upstream and downstream of the dimer. - This action creates a single-stranded gap where the dimer is located. 3. **Removal and Replacement**: - The segment of DNA containing the dimer is excised (removed). - DNA polymerase I then fills in the gap by synthesizing the correct nucleotides, using the undamaged complementary strand as a template. 4. **Sealing the Gap**: - DNA ligase seals the remaining nick in the sugar-phosphate backbone, completing the repair. **Xeroderma Pigmentosa (XP)** - **Condition Overview**: - Individuals with xeroderma pigmentosa (XP) have a genetic defect in the enzymes responsible for nucleotide excision repair. - This results in a heightened sensitivity to UV light and an inability to repair thymine dimers effectively. - **Consequences**: - When exposed to UV radiation, the accumulation of thymine dimers can lead to significant DNA damage. - Affected individuals are at a higher risk of developing skin cancer due to the unrepaired mutations. **Photoreactivation** 1. **Direct Repair Mechanism**: - Photoreactivation is a light-dependent repair process that directly reverses thymine dimers. - An enzyme called photolyase binds to the dimer, recognizing the distortion it causes in the DNA structure. 2. **Light Activation**: - When exposed to visible light, photolyase undergoes a conformational change that allows it to cleave the covalent bond between the two thymine bases. - This restores the correct base pairing, allowing the DNA to function normally. 3. **Organismal Distribution**: - Photoreactivation is found in many organisms, including plants, photosynthetic bacteria, algae, and corals. - However, it is absent in placental mammals, including humans, making other repair mechanisms like nucleotide excision repair critical for our survival. **Importance of Repair Mechanisms** - **Genomic Stability**: Effective repair of thymine dimers is crucial for maintaining genomic integrity, preventing mutations that can lead to cancer and other genetic disorders. - **Adaptation to Environment**: Organisms exposed to high levels of UV radiation, such as those in sunny environments, rely heavily on these repair mechanisms to counteract the potential mutagenic effects of sunlight. Together, nucleotide excision repair and photoreactivation exemplify the biological systems evolved to protect against DNA damage from environmental factors, ensuring the continued survival and health of organisms across different environments. **[5.4 Causes of Mutations and Direct Repair of DNA Damage]** I. **Overview of Transcription** - - - - - - - - - - - - - - - - II. **Transcription in prokaryotes I** - - - - **Importance of the -35 and -10 Regions in the Promoter** - - - - - - - - - - - III. **Transcription in Prokaryotes II** - - - - - - - - - - - IV. **Initiation of Transcription in Prokaryotes** - **Simultaneous Processes**: - Prokaryotes lack membrane-enclosed nuclei, allowing transcription, translation, and mRNA degradation to occur simultaneously. - This enables rapid amplification of bacterial protein levels through concurrent transcription and translation events on the same DNA template. - **Polycistronic mRNAs**: - Prokaryotic transcription can cover multiple genes, producing **polycistronic mRNAs** that code for more than one protein. - While there are differences between transcription in E. coli and archaea, E. coli transcription principles apply broadly to bacterial species. **Prokaryotic RNA Polymerase** - **Single RNA Polymerase**: - Prokaryotes use one RNA polymerase to transcribe all genes. - RNA polymerase adds nucleotides to the 3'-OH group of the growing RNA strand, unlike DNA polymerase, which requires a primer with a 3'-OH. - **Transcription Process**: - Ribonucleotides complementary to the DNA template are added to the growing RNA strand, forming phosphodiester bonds via dehydration synthesis. - **E. coli RNA Polymerase Core**: - Composed of five polypeptide subunits: αI, αII, β, β\', and ω. - The α-subunits assemble polymerase on DNA, the β-subunit binds ribonucleoside triphosphates, and β\' binds the DNA template. - The sigma subunit (σ) binds to the core enzyme, inducing a conformational change that activates it and ensures transcription starts at the correct initiation site. - The active form, comprising the core enzyme and σ, is called the **holoenzyme**. A diagram of a dna polymerase Description automatically generated **Prokaryotic Promoters** - **Promoter Function**: - Promoters are DNA sequences where transcription machinery binds to initiate transcription, usually located upstream of the regulated genes. - The specific sequence of a promoter influences the frequency of transcription. - **Consensus Sequences**: - Promoter sequences vary but often contain conserved elements. - The -10 region (Pribnow box: TATAAT) and the -35 region (TTGACA) are crucial for σ binding and initiation. - The A--T-rich -10 region aids in unwinding the DNA template. - **Transcription Initiation Phase**: - Ends with the production of **abortive transcripts**---short RNA polymers (about 10 nucleotides) that are synthesized and released. V. **Elongation and Termination in Prokaryotes** - - - - - - - - -  VI. **Prokaryotic Termination Signals** - - - - - - - - - - - - - VII. **Transcription in Eukaryotes** - Both prokaryotes and eukaryotes perform transcription, but with significant differences. - **RNA Polymerases**: - **Eukaryotes**: Use three distinct RNA polymerases (I, II, and III), each transcribing different subsets of genes. - **Archaea**: Have a single RNA polymerase more similar to eukaryotic RNA polymerase II than to bacterial RNA polymerase. - **Prokaryotes**: Use one RNA polymerase for all genes. - **mRNA Structure**: - **Eukaryotic mRNAs**: Typically **monocistronic**, encoding a single polypeptide. - **Prokaryotic mRNAs**: Often **polycistronic**, encoding multiple polypeptides. - **Membrane-Bound Nucleus**: - Eukaryotes possess a nucleus, requiring RNA transport from the nucleus to the cytoplasm for translation. - Prokaryotic mRNA degrades rapidly (typically within 5 seconds), while eukaryotic mRNA can last several hours due to protective modifications. **Eukaryotic mRNA Processing** - **Primary Transcript**: - Also known as **pre-mRNA**, is initially coated with RNA-stabilizing proteins to protect against degradation during processing and transport. - **5' Capping**: - A 7-methylguanosine cap is added to the 5' end during transcription to prevent degradation and assist in ribosome recognition for translation initiation. - **3' Poly-A Tail**: - Approximately 200 adenine nucleotides are added to the 3' end post-elongation, further protecting the mRNA and signaling export to the cytoplasm. - **Exons and Introns**: - **Exons**: Coding sequences that are expressed. - **Introns**: Non-coding sequences that are removed from the pre-mRNA during processing. - Proper removal of introns and joining of exons is crucial for producing a functional polypeptide. - **RNA Splicing**: - The process of removing introns and reconnecting exons, facilitated by a **spliceosome** containing small nuclear ribonucleoproteins (**snRNPs**). - Errors in splicing can lead to nonfunctional polypeptides. - **Alternative Splicing**: - Allows different mRNA transcripts to be generated from the same DNA sequence by including or excluding various exons. - This enhances protein diversity from a single gene. - **Intron Functions**: - While not translated, introns may have roles in gene regulation and mRNA transport. - Some archaea also demonstrate the ability to splice their pre-mRNA. Initiation of Transcription in Eukaryotes - **Transcription Factors Requirement**: - Unlike prokaryotic polymerase, which can bind directly to DNA, eukaryotic polymerases require several transcription factors to bind to the promoter region before recruitment. **Three Eukaryotic RNA Polymerases** - **RNA Polymerase I**: - **Location**: Nucleolus. - **Function**: Synthesizes all ribosomal RNA (rRNA) except for 5S rRNA. - **Role of rRNA**: Structural RNA, part of ribosomes, essential for translation. - **Svedberg Units**: "S" designation reflects sedimentation speed during centrifugation. - **RNA Polymerase II**: - **Location**: Nucleus. - **Function**: Synthesizes all protein-coding nuclear pre-mRNAs. - **Processing**: Pre-mRNAs undergo extensive processing before translation. The term \"mRNAs\" refers only to mature, processed molecules ready for translation. - **Significance**: Responsible for transcribing the majority of eukaryotic genes. - **RNA Polymerase III**: - **Location**: Nucleus. - **Function**: Transcribes various structural RNAs, including: - 5S pre-rRNA - Transfer pre-RNAs (pre-tRNAs) - Small nuclear pre-RNAs (snRNAs) - **Role of tRNA**: Serves as adaptor molecules between mRNA and the growing polypeptide chain. - **Role of snRNAs**: Involved in splicing pre-mRNAs and regulating transcription factors. **Sensitivity to α-Amanitin** - **α-Amanitin Effects**: - **RNA Polymerase I**: Completely insensitive; can transcribe DNA in the presence of the poison. - **RNA Polymerase II**: Extremely sensitive; inhibited by α-amanitin. - **RNA Polymerase III**: Moderately sensitive to α-amanitin. - **Research Implications**: Understanding which polymerase transcribes a gene can provide insights into the gene's function. - **Focus on RNA Polymerase II**: Given its role in transcribing the majority of genes, further discussions on eukaryotic transcription will concentrate on RNA polymerase II, its transcription factors, and associated promoters. VIII. **Structure of an RNA Polymerase II Promoter** - **Complexity of Eukaryotic Promoters**: - Eukaryotic promoters are larger and more complex than prokaryotic promoters. - Example: In the mouse thymidine kinase gene, the TATA box is located at approximately -30 relative to the +1 (initiation) site. - TATA box sequence: TATAAA (5\' to 3\' on the nontemplate strand). - Contains an A--T rich element, facilitating local unwinding of DNA for transcription. - **Transcription Initiation**: - **Binding Process**: - Transcription factors first bind to the promoter region. - RNA polymerase II subsequently binds to form the transcription initiation complex. - **Introns and mRNA Processing**: - Eukaryotic mRNA contains introns that must be spliced out. - A 5\' cap and a 3\' poly-A tail are added to the mRNA during processing. - **Thymidine Kinase Gene and Pseudogenes**: - The mouse genome includes one functional gene and two pseudogenes for cytoplasmic thymidine kinase. - **Pseudogenes**: Genes that have lost their protein-coding ability or are no longer expressed; they are derived from mRNA and incorporated into the chromosome. - **Additional Promoter Elements**: - The mouse thymidine kinase promoter contains: - **CAAT box**: Located at approximately -80, with the sequence GGCCAATCT; essential for transcription factor binding. - **GC-rich boxes (GGCG)** and **octamer boxes (ATTTGCAT)**: Found further upstream; these elements bind cellular factors to enhance transcription initiation and are prevalent in actively expressed genes. **Promoter Differences for RNA Polymerases** - **RNA Polymerase I**: - Transcribes genes with two GC-rich promoter sequences located in the -45 to +20 region, sufficient for transcription initiation. - Additional sequences in the -180 to -105 region enhance initiation. - **RNA Polymerase III**: - Genes transcribed by RNA polymerase III have upstream promoters or promoters located within the genes themselves. IX. **Transcription Factors for RNA Polymerase II** **1. Regulatory Elements** - **Enhancers**: - **Function**: Increase the efficiency of transcription. - **Nature**: Not essential for transcription but significantly enhance its rate. - **Silencers**: - **Function**: Decrease transcription efficiency. - **Nature**: Similar to enhancers, they modulate transcription but are not required for it. **2. Basal Transcription Factors** - **Role**: Essential for forming the preinitiation complex at the promoter region. - **Naming**: Basal transcription factors for RNA polymerase II are designated as \"TFII\" (e.g., TFIIA, TFIIB, etc.). - **Functionality**: - Bind in a specific order to stabilize the preinitiation complex. - Facilitate the recruitment of RNA polymerase II, enabling transcription initiation. **3. Transcription Factors for RNA Polymerases I and III** - Involve fewer and less complex transcription factors compared to RNA polymerase II. - Overall principle remains similar, focusing on recruitment for transcription initiation. **4. Overall Complexity and Investment** - **Metabolic Investment**: Eukaryotic transcription requires more energy and resources than prokaryotic transcription due to numerous regulatory proteins and processes. - **Precision in Gene Expression**: This complexity allows eukaryotic cells to finely tune the transcription of pre-mRNAs, ensuring appropriate protein synthesis as needed. **Summary** Eukaryotic transcription involves intricate interactions between various transcription factors, enhancers, and silencers, ensuring precise and regulated expression of genes. This complexity is essential for the sophisticated control required for diverse eukaryotic cell functions. X. **Elongation and Termination in Eukaryotes** **Elongation Process** - **Preinitiation Complex**: - RNA polymerase II is released from transcription factors to begin elongation. - **Direction**: - RNA polymerase II synthesizes pre-mRNA in the 5\' to 3\' direction, similar to prokaryotes. - **Chromatin Structure**: - Eukaryotic DNA is organized into chromatin, complicating transcription due to coiling around histone proteins into nucleosomes. - **Role of FACT Complex**: - **FACT (Facilitates Chromatin Transcription)**: - Displaces histones as RNA polymerase II encounters nucleosomes to access the DNA template. - Replaces histones after RNA polymerase II passes to maintain chromatin structure. **2. Termination Process** - **Polymerase II Termination**: - RNA polymerase II often transcribes 1,000--2,000 nucleotides beyond the gene, forming a pre-mRNA tail that is cleaved during processing. - **Polymerases I and III Termination**: - **RNA Polymerase I**: - Recognizes a specific 18-nucleotide termination sequence bound by a termination protein to signal transcription\'s end. - **RNA Polymerase III**: - Utilizes a mechanism similar to rho-independent termination in prokaryotes. - Forms an mRNA hairpin structure that causes the polymerase to stall and detach from the DNA template. **Summary** - The elongation phase of eukaryotic transcription is facilitated by the FACT complex, allowing RNA polymerase II to navigate chromatin effectively. - Termination processes differ among the three eukaryotic polymerases: - RNA polymerase II extends beyond the gene before mRNA processing. - Polymerases I and III rely on specific sequences and structural cues for termination. - This intricate regulation ensures precise control over gene expression in eukaryotic cells. XI. **mRNA Processing in Eukaryotes** - Eukaryotic mRNAs last for several hours. - Typical E. coli mRNA lasts no more than five seconds. - Pre-mRNAs are coated in RNA-stabilizing proteins to prevent degradation during processing and nuclear export. 1. **5\' Capping**: - A 7-methylguanosine cap is added to the 5\' end via a phosphate linkage. - This cap protects the mRNA from degradation and aids in translation initiation by ribosomes. 2. **3\' Poly-A Tail**: - Pre-mRNA is cleaved by an endonuclease downstream of the AAUAAA consensus sequence. - Poly-A polymerase adds approximately 200 adenine (A) residues, forming a poly-A tail. - This modification further protects the mRNA and signals export to the cytoplasm. 3. **Pre-mRNA Splicing**: - Eukaryotic genes consist of: - - - Introns may play roles in gene regulation but do not encode functional proteins. - Found unexpectedly in the 1970s; researchers anticipated pre-mRNAs would directly specify proteins without processing. - Higher eukaryotes often contain multiple introns, raising questions about their biological significance. - They may slow down gene expression or be remnants of ancient genes. - Introns can often be mutated without affecting the protein product. - All introns must be accurately removed before protein synthesis. - Errors in splicing can shift the reading frame of rejoined exons, leading to dysfunctional proteins. - **Splicing**: The process of removing introns and reconnecting exons. - Conducted by **spliceosomes**, which are complexes of proteins and RNA. - XII. **Processing of tRNA and rRNAs** **1. Structural Role** - **tRNAs and rRNAs**: - Serve as structural molecules in protein synthesis but are not translated into proteins. **2. Processing and Assembly** - **Pre-rRNAs**: - Transcribed, processed, and assembled into ribosomes in the nucleolus. - **Pre-tRNAs**: - Transcribed and processed in the nucleus. - Released into the cytoplasm, where they are linked to free amino acids for protein synthesis. **3. Precursor Molecules** - Most tRNAs and rRNAs are initially transcribed as long precursor molecules that encompass multiple rRNAs or tRNAs. - Enzymes cleave these precursors into individual subunits for each structural RNA. **4. Methylation** - Some bases in pre-rRNAs are methylated (addition of a --CH3 moiety), enhancing stability. - Pre-tRNAs also undergo methylation. **5. Ribosomal Composition** - Mature rRNAs constitute approximately 50% of each ribosome. - Ribosomal RNA includes: - **Structural RNAs**: Provide the framework of the ribosome. - **Catalytic or Binding Activities**: Facilitate biochemical processes. **6. tRNA Structure** - Mature tRNAs achieve a three-dimensional structure via intramolecular hydrogen bonding. - Key features: - **Amino Acid Binding Site**: Located at one end of the tRNA. - **Anticodon**: A three-nucleotide sequence at the opposite end that pairs with the mRNA codon through complementary base pairing. **[5.6 Translation]** I. **Gene Structure** **1. Definition and Function** - **Gene Structure**: Organization of specialized sequence elements within a gene. - **Purpose**: Contains information necessary for living cells to survive and reproduce. - **Composition**: Genes are primarily made of DNA, with specific sequences determining gene function. **2. Transcription and Translation** - **Transcription**: Genes are copied from DNA into RNA. - **Types of RNA**: - **Non-coding RNA (ncRNA)**: Has a direct function. - **Messenger RNA (mRNA)**: Translated into protein. - **Control Mechanism**: Each transcription and translation step is regulated by specific sequence elements within the gene. **3. Sequence Elements** - **Functional Requirement**: Each gene requires multiple sequence elements: - **Coding Sequence**: Encodes functional proteins or ncRNA. - **Regulatory Sequence Regions**: Can vary in length from a few base pairs to thousands. **4. Similarities and Differences** - **Eukaryotes vs. Prokaryotes**: Gene structures are broadly similar, but key differences arise due to their distinct transcription and translation machinery. **5. Nested Sequence Elements** - **Structure**: Both eukaryotic and prokaryotic genes involve several nested elements, each with specific functions in gene expression. - **Sense Strand**: Typically, only one strand of DNA (the \'sense\' or \'coding\' strand) is read by RNA polymerase to produce RNA. **6. Regulatory Sequences** - **Location**: Found at the extremities of genes. - **Promoter**: Located at the 5\' end, includes: - **Core Promoter Sequence** - **Proximal Promoter Sequence** - **Enhancers and Silencers**: Can be located far from the transcribed region. **7. Untranslated Regions (UTRs)** - **Protein-Coding Genes**: The region between start and stop codons encodes the protein. - **5\' UTR**: Region before the coding sequence; binds the ribosome for translation. - **3\' UTR**: Follows the coding region; contains the terminator sequence that marks the end of transcription and releases RNA polymerase. **8. Additional Information** - **Open Reading Frame (ORF)**: Represents the sequence that is read to produce the protein, usually shown as an arrow indicating the reading direction of the sense strand. II. **The Genetic Code** - **Translation Process**: Converts mRNA\'s nucleotide sequence into a sequence of amino acids, forming a protein. - **Amino Acids**: Composed of 20 commonly occurring amino acids. - **Codons**: - Defined as triplets of nucleotides in mRNA. - Each codon specifies a particular amino acid. - **Genetic Code**: - Total of 64 possible codons (4 nucleotides in triplet combinations: 43=644\^3 = 6443=64). - More codons than amino acids; thus, most amino acids are specified by more than one codon, a phenomenon known as **degeneracy**. - **Wobble Position**: - The third position in a codon is less critical for determining the incorporated amino acid. - Changes in the third nucleotide often do not affect the resulting amino acid. - **Codons and Their Functions**: - **61 codons** specify amino acids. - **3 codons** are stop codons (UAA, UAG, UGA) that terminate protein synthesis. - **AUG** serves as both a start codon and specifies the amino acid methionine. - **Reading Frame**: - Set by the AUG start codon located near the 5\' end of mRNA. - Subsequent sets of three nucleotides are read as codons. - **Universality of the Genetic Code**: - Nearly universal among species, indicating a common origin of life. - Exceptions include unusual amino acids like selenocysteine and pyrrolysine. - **Selenocysteine**: - Encoded by the UGA codon (normally a stop codon) when a specific mRNA structure (SECIS element) is present. - **Pyrrolysine**: - Uses the UAG codon as a stop codon but can incorporate pyrrolysine with the help of the pylS gene and a specific tRNA (with a CUA anticodon). **Summary** Translation is a critical step in gene expression, converting the nucleotide sequences of mRNA into functional proteins through codons. The genetic code is nearly universal and contains redundancies that allow for variations in codon usage, with special mechanisms for the incorporation of uncommon amino acids like selenocysteine and pyrrolysine. III. **Amino Acids** - **Fundamental Structure**: - Central carbon atom (α carbon) bonded to: - An amino group (NH₂) - A carboxyl group (COOH) - A hydrogen atom - An R group (side chain), which varies for each amino acid - **Name Origin**: - \"Amino acid\" reflects the presence of both an amino group and a carboxyl group. - **Common Amino Acids**: - There are **20 common amino acids** found in proteins. - Each amino acid has a unique R group that defines its chemical nature. - **Essential Amino Acids**: - Out of the 20, **10 are considered essential** for humans, meaning they must be obtained from the diet because the body cannot synthesize them. - **Chemical Nature of R Groups**: - Determines the classification of amino acids: - **Nonpolar (Hydrophobic)**: Valine, Methionine, Alanine - **Polar (Hydrophilic)**: Serine, Threonine, Cysteine - **Basic (Positively Charged)**: Lysine, Arginine - **Unique Structure**: Proline has a ring structure formed by the R group connecting back to the amino group, making it an exception. - **Amino Acid Representation**: - Amino acids can be represented by: - A single uppercase letter (e.g., Valine = V) - A three-letter abbreviation (e.g., Valine = Val) - **Essential Amino Acids in Humans**: - Include isoleucine, leucine, and cysteine, among others. - Essential amino acids are crucial for protein synthesis but are not produced by the body. - **Protein Characteristics**: - The **sequence and number of amino acids** in a protein determine its shape, size, and function. **Summary** Amino acids are the building blocks of proteins, each with a unique structure defined by its R group. The properties of these side chains determine the classification of amino acids as nonpolar, polar, or basic. Essential amino acids must be obtained from the diet, and the specific sequence of amino acids in proteins dictates their ultimate structure and function. IV. **Protein Synthesis** - **Energy Consumption**: - Protein synthesis consumes more energy than any other metabolic process in cells. - Proteins account for more mass in living organisms than any other component (except water). - Proteins perform virtually every cellular function. - **Translation Process**: - Translation involves decoding an mRNA message into a polypeptide product. - Amino acids are covalently linked by peptide bonds, forming polypeptides of varying lengths (50 to over 1,000 amino acid residues). - **Amino Acid Structure**: - Each amino acid contains: - An amino group (NH₂) - A carboxyl group (COOH) - **Peptide Bond Formation**: - A peptide bond forms between the amino group of one amino acid and the carboxyl group of another, resulting in the release of one water molecule. - This reaction is catalyzed by ribosomes. - Functional groups involved in peptide bond formation include the amino and carboxyl groups of the respective amino acids. - **Polypeptide Structure**: - Each polypeptide has: - A free amino group at one end (N terminal or amino terminal) - A free carboxyl group at the other end (C terminal or carboxyl terminal) - **Terminology**: - **Polypeptide**: A polymer of amino acids. - **Protein**: A polypeptide or multiple polypeptides that are often combined, have bound non-peptide prosthetic groups, distinct shapes, and unique functions. - **Post-Translational Modifications**: - After synthesis, most proteins undergo modifications, including: - Cleavage - Phosphorylation - Addition of other chemical groups - These modifications are crucial for the protein\'s full functionality. **Summary** Protein synthesis is a complex and energy-intensive process that converts mRNA into functional proteins through the formation of peptide bonds between amino acids. The resulting polypeptides undergo various post-translational modifications to become fully functional proteins. V. **Protein Synthesis Machinery: Ribosomes** - **Composition**: - Ribosomes are macromolecules made of: - Catalytic rRNAs (ribozymes) - Structural rRNAs - Distinct polypeptides - Mature rRNAs constitute approximately 50% of each ribosome. - **Ribosome Types**: - **Prokaryotic Ribosomes**: - Size: 70S - Small subunit: 30S (contains 16S rRNA) - Large subunit: 50S (contains 5S and 23S rRNA) - **Eukaryotic Ribosomes**: - Size: 80S - Small subunit: 40S (contains 18S rRNA) - Large subunit: 60S (contains 5S, 5.8S, and 28S rRNA) - Ribosomes in mitochondria and chloroplasts are 70S. - **Subunit Dynamics**: - Ribosomes dissociate into large and small subunits when not synthesizing proteins. - Subunits reassociate during translation initiation. - **Translation Process**: - Multiple ribosomes can simultaneously translate a single mRNA molecule, forming a **polyribosome (or polysome)**. - Translation occurs from 5' to 3' on mRNA, synthesizing the polypeptide from N terminus to C terminus. - **Simultaneous Transcription and Translation**: - In **Bacteria and Archaea**: - Transcription and translation can occur concurrently because: - Both processes proceed in the same 5' to 3' direction. - Both occur in the cytoplasm. - The RNA transcript is not processed post-transcription. - This allows rapid protein synthesis in response to environmental signals. - In **Eukaryotes**: - Simultaneous transcription and translation is not possible due to the compartmentalization of the nucleus. - Polyribosomes can only form after RNA synthesis is complete and the RNA has been modified and transported out of the nucleus. **Summary** Ribosomes are essential macromolecular complexes composed of rRNAs and proteins, differing in size between prokaryotes and eukaryotes. They play a crucial role in translating mRNA into proteins, with multiple ribosomes often translating a single mRNA simultaneously, forming polyribosomes. In prokaryotes, transcription and translation occur concurrently, while in eukaryotes, they are sequential due to nuclear compartmentalization. VI. **Protein Synthesis Machinery: Transfer RNAs (tRNAs)** - **Definition**: - tRNAs are structural RNA molecules that serve as adaptors in the translation process. - **Variety in Species**: - Bacterial species typically have between 60 and 90 different types of tRNAs. - **Role in Translation**: - Each tRNA binds to a specific codon on the mRNA and adds the corresponding amino acid to the growing polypeptide chain. - tRNAs effectively "translate" the RNA language into the protein language. - **Interactions**: - tRNAs interact with three key factors: - **Aminoacyl-tRNA synthetases**: Enzymes that attach the correct amino acid to the tRNA. - **Ribosomes**: The cellular machinery where protein synthesis occurs. - **mRNA**: The messenger RNA template that provides the codon sequence. - **Three-Dimensional Structure**: - Mature tRNAs adopt a specific three-dimensional shape through intramolecular hydrogen bonding among complementary bases in the single-stranded RNA. - This structure positions: - The **CCA amino acid binding end** (a cytosine-cytosine-adenine sequence at the 3' end). - The **anticodon**, which is a three-nucleotide sequence that pairs with an mRNA codon. - **tRNA Charging Process**: - Each tRNA is linked to its corresponding amino acid through a process called tRNA \"charging.\" - **Aminoacyl-tRNA synthetases** activate the amino acid by adding adenosine monophosphate (AMP) before transferring it to the tRNA. - This results in a charged tRNA, with AMP released during the process. **Summary** Transfer RNAs (tRNAs) are crucial adaptors in protein synthesis, translating mRNA codons into amino acids. They possess a specific structure that allows them to interact with ribosomes and aminoacyl-tRNA synthetases, ensuring the correct amino acid is added to the growing polypeptide chain through a process known as tRNA charging. VII. **Initiation of Translation** **In E. coli:** - **Initiation Complex Components**: - Small 30S ribosomal subunit. - mRNA template. - Three initiation factors (IFs): IF-1, IF-2, IF-3. - Guanosine triphosphate (GTP) as an energy source. - Special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet). - **Initiator tRNA Function**: - fMet-tRNAfMet interacts with the start codon AUG on the mRNA and carries formylated methionine (fMet) to the P site. - fMet is inserted at the N-terminus of every polypeptide synthesized by E. coli. - **Shine-Dalgarno Sequence**: - A leader sequence upstream of the first AUG codon. - Known as the ribosomal binding site (AGGAGGU), it pairs with rRNA in the ribosome, anchoring the 30S subunit at the correct location on the mRNA. - **Formation of the Complete Ribosome**: - The 50S ribosomal subunit binds to the initiation complex, forming an intact ribosome. **In Eukaryotes:** - **Initiation Complex Components**: - 40S small ribosomal subunit. - mRNA. - Initiation factors (IFs). - Nucleoside triphosphates (GTP and ATP). - Charged initiator tRNA called Met-tRNAi. - **Binding Mechanism**: - Unlike E. coli, there is no Shine-Dalgarno sequence; instead, a cap-binding protein (CBP) and IFs assist the ribosome\'s movement to the 5\' cap of the mRNA. - The complex tracks along the mRNA in the 5' to 3' direction until it recognizes the AUG start codon. - **Kozak\'s Rules**: - Eukaryotic mRNAs are often translated from the first AUG, but specific sequences around the AUG influence its recognition. - Consensus sequence around the AUG in vertebrate genes: 5\'-A/GccAUGG-3\'. - Closer adherence to this sequence increases translation efficiency. - **Completion of Initiation**: - Once the correct AUG is identified, other proteins and CBP dissociate. - The 60S ribosomal subunit binds to the Met-tRNAi, mRNA, and 40S subunit, completing translation initiation in eukaryotes. VIII. **Elongation** **Key Ribosomal Sites in E. coli:** - **A (Aminoacyl) Site**: - Binds incoming charged aminoacyl-tRNAs. - **P (Peptidyl) Site**: - Binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain, without dissociating from their tRNA. - **E (Exit) Site**: - Releases dissociated tRNAs for recharging with free amino acids. **Special Consideration:** - During initiation, fMet-tRNAfMet (in prokaryotes) or Met-tRNAi (in eukaryotes) enters directly into the P site, leaving the A site free for the first codon after AUG. **Elongation Process:** - **Translocation Events**: - Ribosome moves by single codons (three bases) during each translocation event. - **tRNA Movement**: - Charged tRNAs enter the A site, shift to the P site, and then move to the E site for removal. - **Ribosomal Movement**: - Induced by conformational changes, advancing the ribosome in the 3' direction. - **Peptide Bond Formation**: - Catalyzed by peptidyl transferase (an RNA-based ribozyme in the 50S subunit). - Forms between the amino group of the amino acid in the A site and the carboxyl group of the amino acid in the P site. - **Polypeptide Chain Growth**: - The amino acid in the P site is linked to the growing polypeptide chain. - **tRNA Release**: - Former P-site tRNA enters the E site, detaches from its amino acid, and is expelled. **Energy Requirement:** - Several steps in elongation, including tRNA binding and translocation, require energy from GTP hydrolysis, catalyzed by specific elongation factors. **Speed of Translation:** - E. coli can add each amino acid in approximately 0.05 seconds, allowing for the translation of a 200-amino-acid protein in just 10 seconds. IX. **Termination** - - - - - **Protein Folding, Modification, and Targeting** - - - - - - - - A diagram of a protein structure Description automatically generated X. **Translation in Prokaryotes vs. Eukaryotes**