Structure and Function of DNA and RNA

DNA and RNA: Review Questions:

Question 1:
What are the main structural differences between DNA and RNA?

Answer: DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) are both nucleic acids but differ in several key structural aspects:

1. Sugar Component:

   • DNA: Contains deoxyribose sugar, which lacks an oxygen atom at the 2′ carbon.
   • RNA: Contains ribose sugar, which has a hydroxyl (-OH) group at the 2′ carbon.

2. Nitrogenous Bases:

   • DNA: Uses adenine (A), thymine (T), cytosine (C), and guanine (G).
   • RNA: Uses adenine (A), uracil (U) instead of thymine, cytosine (C), and guanine (G).

3. Strand Structure:

   • DNA: Typically double-stranded, forming a stable double helix.
   • RNA: Usually single-stranded, allowing it to fold into various shapes.

4. Function and Stability:

   • DNA: Primarily serves as the long-term storage of genetic information; more stable due to the lack of the 2′ hydroxyl group.
   • RNA: Involved in protein synthesis and other cellular functions; less stable because the 2′ hydroxyl group makes it more prone to hydrolysis.

5. Length:

   • DNA: Generally much longer, containing the complete genetic blueprint of an organism.
   • RNA: Shorter in length, often transient, serving specific roles like messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).

These structural differences underpin the distinct roles DNA and RNA play in cellular biology.

Question 2:
Describe the process of DNA replication, including the roles of key enzymes involved.

Answer: DNA replication is a fundamental process ensuring that each cell inherits an identical copy of the organism’s genome. It occurs during the S-phase of the cell cycle and involves several key steps and enzymes:

1. Initiation:

   • Origin of Replication: Specific sequences where replication begins.
   • Helicase: Unwinds the double helix by breaking hydrogen bonds between base pairs, creating a replication fork.

2. Primer Binding:

   • Primase: Synthesizes short RNA primers complementary to the DNA template, providing a starting point for DNA synthesis.

3. Elongation:

   • DNA Polymerase III (in prokaryotes) or DNA Polymerase δ and ε (in eukaryotes): Adds new complementary nucleotides to the 3′ end of the RNA primer, synthesizing the new DNA strand in the 5′ to 3′ direction.
   • Leading Strand Synthesis: Synthesized continuously towards the replication fork.
   • Lagging Strand Synthesis: Synthesized discontinuously away from the replication fork in short fragments called Okazaki fragments.

4. Primer Replacement and Ligation:

   • DNA Polymerase I (in prokaryotes) or RNase H and DNA Polymerase δ (in eukaryotes): Removes RNA primers and replaces them with DNA nucleotides.
   • DNA Ligase: Seals the nicks between Okazaki fragments, forming a continuous DNA strand.

5. Termination:

   • Telomerase (in eukaryotes): Extends the ends of linear chromosomes (telomeres) to prevent loss of genetic information during replication.

Key Enzymes:

• Helicase: Unwinds the DNA double helix.
• Primase: Synthesizes RNA primers.
• DNA Polymerases: Add nucleotides to the growing DNA strand.
• Ligase: Connects DNA fragments.
• Topoisomerase: Prevents supercoiling ahead of the replication fork by cutting and rejoining DNA strands.

DNA replication is highly accurate, thanks to proofreading activity of DNA polymerases and mismatch repair mechanisms that correct any errors that escape initial replication fidelity.

Question 3:
Explain the role of messenger RNA (mRNA) in protein synthesis.

Answer: Messenger RNA (mRNA) plays a crucial role in the process of protein synthesis, serving as the intermediary between the genetic information encoded in DNA and the synthesis of proteins by ribosomes. Its role can be outlined in the following steps:

1. Transcription:

   • Synthesis: mRNA is synthesized from a DNA template in the nucleus through the process of transcription, where RNA polymerase binds to the promoter region of a gene and transcribes the DNA sequence into a complementary mRNA strand.
   • Processing (in eukaryotes): The primary mRNA transcript undergoes splicing to remove introns, addition of a 5′ cap, and polyadenylation at the 3′ end to form mature mRNA.

2. Export:

   • Transportation: Mature mRNA is transported from the nucleus to the cytoplasm through nuclear pores.

3. Translation:

   • Ribosome Binding: mRNA binds to ribosomes, the molecular machines that facilitate protein synthesis.
   • Codon Recognition: The ribosome reads the mRNA sequence in sets of three nucleotides called codons, each specifying a particular amino acid.
   • tRNA Interaction: Transfer RNA (tRNA) molecules, each carrying a specific amino acid, recognize and bind to the corresponding codon on the mRNA through their anticodon regions.
   • Peptide Bond Formation: The ribosome catalyzes the formation of peptide bonds between adjacent amino acids, elongating the polypeptide chain.
   • Termination: Once a stop codon is reached, the ribosome releases the completed protein.

Summary: mRNA serves as the template that conveys genetic information from DNA to the ribosomes, dictating the order in which amino acids are assembled to form proteins. This process ensures that proteins are synthesized accurately according to the genetic instructions, enabling cellular functions and contributing to the organism’s phenotype.

Question 4:
What are ribosomal RNA (rRNA) molecules, and what is their function in the ribosome?

Answer: Ribosomal RNA (rRNA) molecules are essential components of ribosomes, the cellular structures responsible for protein synthesis. rRNAs play both structural and catalytic roles within the ribosome, facilitating the translation of mRNA into proteins.

Types of rRNA:

• Prokaryotes:

  • 16S rRNA: Part of the small (30S) ribosomal subunit; involved in mRNA recognition and initiation of translation.
  • 23S rRNA: Part of the large (50S) ribosomal subunit; contains the peptidyl transferase center responsible for peptide bond formation.
  • 5S rRNA: Also part of the large (50S) subunit; contributes to the structural stability of the ribosome.

• Eukaryotes:

  • 18S rRNA: Part of the small (40S) ribosomal subunit; analogous to prokaryotic 16S rRNA.
  • 28S rRNA: Part of the large (60S) subunit; analogous to prokaryotic 23S rRNA.
  • 5.8S rRNA: Also part of the large (60S) subunit; works in conjunction with 28S and 5S rRNAs.
  • 5S rRNA: Present in both prokaryotes and eukaryotes, part of the large subunit.

Functions of rRNA in the Ribosome:

1. Structural Role:

   • rRNAs form the scaffold of the ribosome, maintaining its three-dimensional structure and ensuring proper alignment of mRNA and tRNA during translation.

2. Catalytic Role:

   • Peptidyl Transferase Activity: The 23S (prokaryotes) or 28S (eukaryotes) rRNA catalyzes the formation of peptide bonds between amino acids, a critical step in protein elongation. This makes rRNA a ribozyme, an RNA molecule with catalytic activity.

3. mRNA and tRNA Binding:

   • Decoding Center: rRNA interacts with mRNA and tRNA at the decoding center, ensuring accurate base-pairing between codons and anticodons, which is vital for the fidelity of protein synthesis.
   • A, P, and E Sites: rRNA helps form the A (aminoacyl), P (peptidyl), and E (exit) sites within the ribosome, coordinating the entry, bonding, and exit of tRNA molecules during translation.

4. Initiation and Termination:

   • rRNA plays roles in the initiation of translation by facilitating the assembly of the ribosomal subunits with mRNA and initiator tRNA.
   • During termination, rRNA helps recognize stop codons and release the newly synthesized polypeptide chain.

Summary: rRNA molecules are indispensable for ribosome function, providing both the structural framework and the catalytic machinery necessary for translating genetic information into functional proteins. Their dual roles ensure the efficiency and accuracy of protein synthesis within the cell.

Question 5:
Describe the process of transcription in eukaryotic cells, including the key stages and regulatory elements involved.

Answer: Transcription in eukaryotic cells is the process by which genetic information encoded in DNA is copied into messenger RNA (mRNA). This process involves several key stages and regulatory elements to ensure accurate and controlled gene expression.

Stages of Transcription:

1. Initiation:

   • Promoter Recognition: Transcription begins at specific DNA sequences called promoters, located upstream of the coding region. The core promoter includes the TATA box, a conserved sequence typically located about 25-35 base pairs upstream of the transcription start site.
   • Transcription Factors Binding: General transcription factors (e.g., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) assemble at the promoter. TFIID binds to the TATA box via the TATA-binding protein (TBP), initiating the formation of the transcription pre-initiation complex (PIC).
   • RNA Polymerase II Recruitment: TFIID recruits RNA Polymerase II (Pol II) to the promoter, positioning it correctly for transcription to begin.
   • Promoter Melting: TFIIH, which has helicase activity, unwinds the DNA double helix at the promoter, exposing the template strand for transcription.

2. Elongation:

   • RNA Synthesis: RNA Polymerase II moves along the DNA template strand in the 3′ to 5′ direction, synthesizing a complementary RNA strand in the 5′ to 3′ direction by adding ribonucleotides.
   • RNA Processing Co-transcriptionally: As the nascent RNA is being synthesized, it undergoes several processing steps:
     • 5′ Capping: Addition of a 7-methylguanosine cap to the 5′ end, protecting the RNA from degradation and aiding in ribosome binding during translation.
     • Splicing: Removal of non-coding introns and joining of exons by the spliceosome, resulting in a mature mRNA transcript.
     • 3′ Polyadenylation: Addition of a poly-A tail to the 3′ end, enhancing mRNA stability and export from the nucleus.

3. Termination:

   • Termination Signal Recognition: Transcription continues until RNA Polymerase II encounters specific termination signals, often a polyadenylation signal sequence (AAUAAA) in the DNA.
   • RNA Release: Following cleavage at the polyadenylation site, RNA Polymerase II dissociates from the DNA template, releasing the newly synthesized pre-mRNA.
   • Post-Transcriptional Modifications: The pre-mRNA undergoes final processing to become mature mRNA, which is then exported to the cytoplasm for translation.

Regulatory Elements Involved:

1. Enhancers and Silencers:

   • Enhancers: DNA sequences that increase transcription levels when bound by specific transcription factors, often located far from the promoter.
   • Silencers: DNA sequences that decrease transcription levels when bound by repressive transcription factors.

2. Insulators:

   • Function: Act as boundaries to prevent the influence of enhancers and silencers from one gene on another, ensuring proper gene regulation.

3. Promoter Elements:

   • Core Promoter: Contains the TATA box and other elements necessary for the assembly of the transcription machinery.
   • Proximal Promoter Elements: Located near the core promoter, these include binding sites for specific transcription factors that regulate gene expression.

4. Transcription Factors:

   • General Transcription Factors: Required for the initiation of transcription by RNA Polymerase II.
   • Specific Transcription Factors: Bind to enhancers, silencers, or other regulatory elements to modulate transcription levels in response to cellular signals.

Summary: Transcription in eukaryotic cells is a highly regulated process involving the coordinated action of RNA Polymerase II, transcription factors, and various regulatory DNA elements. This ensures that genes are expressed at the right time, in the right cells, and in appropriate amounts, maintaining cellular function and organismal development.

Question 6:
What is the role of transfer RNA (tRNA) in translation, and how does it ensure the correct amino acid is added to the growing polypeptide chain?

Answer: Transfer RNA (tRNA) plays a critical role in the process of translation, where the genetic code carried by messenger RNA (mRNA) is decoded to synthesize proteins. tRNA serves as the adaptor molecule that translates the nucleotide sequence of mRNA into the corresponding amino acid sequence of a protein.

Roles of tRNA in Translation:

1. Amino Acid Transport:

   • Function: Each tRNA molecule carries a specific amino acid to the ribosome, where it is incorporated into the growing polypeptide chain.
   • Aminoacylation: The attachment of an amino acid to its corresponding tRNA is catalyzed by enzymes called aminoacyl-tRNA synthetases. This process ensures that each tRNA is loaded with the correct amino acid.

2. Codon Recognition:

   • Anticodon Loop: tRNA molecules contain a set of three nucleotides called the anticodon, which is complementary to a specific codon on the mRNA.
   • Base Pairing: During translation, the anticodon of the tRNA base-pairs with the appropriate codon on the mRNA within the ribosome’s decoding center. This ensures that the correct amino acid is added according to the genetic code.

3. Positioning within the Ribosome:

   • A Site (Aminoacyl Site): The incoming tRNA carrying an amino acid binds to the A site of the ribosome, matching its anticodon with the mRNA codon.
   • P Site (Peptidyl Site): The tRNA holding the growing polypeptide chain is located in the P site. The amino acid from the tRNA in the A site is transferred to the polypeptide chain, forming a peptide bond.
   • E Site (Exit Site): After the amino acid is added, the now uncharged tRNA moves to the E site and exits the ribosome.

Ensuring Correct Amino Acid Addition:

1. Aminoacyl-tRNA Synthetases Specificity:

   • Each aminoacyl-tRNA synthetase is specific to one amino acid and its corresponding set of tRNAs (isoacceptors). This enzyme ensures that the correct amino acid is attached to the appropriate tRNA, maintaining the fidelity of protein synthesis.

2. Codon-Anticodon Complementarity:

   • The precise base-pairing between the mRNA codon and the tRNA anticodon ensures that each amino acid is added in the correct order as dictated by the mRNA sequence.

3. Proofreading Mechanisms:

   • Aminoacyl-tRNA synthetases have proofreading abilities to detect and correct mischarged tRNAs, further ensuring the accuracy of amino acid incorporation.

Summary: tRNA molecules are essential for translating the genetic code into functional proteins. By carrying specific amino acids and recognizing corresponding mRNA codons through their anticodons, tRNAs ensure that proteins are synthesized accurately and efficiently, reflecting the genetic instructions encoded in the DNA.

Question 7:
What mechanisms regulate gene expression at the transcriptional and post-transcriptional levels in eukaryotic cells?

Answer: Gene expression in eukaryotic cells is tightly regulated at multiple levels to ensure that genes are expressed in the right cells, at the right times, and in appropriate amounts. The regulation occurs both at the transcriptional level (before RNA is synthesized) and post-transcriptional level (after RNA is synthesized).

Transcriptional Regulation:

1. Promoter Accessibility:

   • Chromatin Remodeling: The structure of chromatin (DNA wrapped around histone proteins) can be altered to either expose or hide promoters from the transcription machinery. Euchromatin is open and transcriptionally active, while heterochromatin is condensed and transcriptionally silent.
   • Histone Modifications: Chemical modifications (e.g., acetylation, methylation) of histone tails can influence chromatin structure and gene accessibility.

2. Transcription Factors:

   • General Transcription Factors: Required for the initiation of transcription by RNA Polymerase II.
   • Specific Transcription Factors: Bind to enhancer or silencer regions to increase or decrease the transcription of target genes.

3. Enhancers and Silencers:

   • Enhancers: DNA elements that increase transcription levels when bound by activator proteins.
   • Silencers: DNA elements that decrease transcription levels when bound by repressor proteins.

4. Mediator Complex:

   • Function: Acts as a bridge between transcription factors bound to enhancers/silencers and the RNA Polymerase II machinery, facilitating efficient transcription initiation.

5. DNA Methylation:

   • Role: Addition of methyl groups to cytosine residues in DNA, particularly at CpG islands in promoter regions, generally represses gene transcription by blocking transcription factor binding or recruiting repressive proteins.

6. Non-Coding RNAs:

   • Function: Certain non-coding RNAs can influence transcription by interacting with chromatin-modifying complexes or transcription factors.

Post-Transcriptional Regulation:

1. RNA Splicing:

   • Alternative Splicing: The same pre-mRNA can be spliced in different ways to produce multiple mRNA variants, leading to different protein isoforms from a single gene.

2. RNA Editing:

   • Modification: Nucleotide sequences in the pre-mRNA can be altered post-transcriptionally, changing the coding potential or regulatory elements of the mRNA.

3. mRNA Transport and Localization:

   • Nuclear Export: Regulation of which mRNAs are exported from the nucleus to the cytoplasm can influence gene expression levels.
   • mRNA Localization: Specific mRNAs can be transported to particular regions within the cell for localized protein synthesis.

4. mRNA Stability and Degradation:

   • Regulatory Elements: Sequences in the mRNA (e.g., AU-rich elements) can influence its stability and half-life.
   • MicroRNAs (miRNAs) and siRNAs: These small non-coding RNAs can bind to complementary sequences on mRNAs, leading to mRNA degradation or inhibition of translation.

5. Translation Regulation:

   • Initiation Factors: Control the assembly of the ribosome on the mRNA, affecting the rate of protein synthesis.
   • Upstream Open Reading Frames (uORFs): Regulatory sequences in the 5′ untranslated region (UTR) can modulate translation efficiency.

6. Post-Translational Modifications:

   • Protein Modifications: Although occurring after translation, modifications like phosphorylation, ubiquitination, and glycosylation can regulate protein activity, stability, and localization, indirectly influencing gene expression outcomes.

Summary: Gene expression in eukaryotic cells is orchestrated through a complex interplay of regulatory mechanisms at both the transcriptional and post-transcriptional levels. These mechanisms ensure precise control over which genes are expressed, when, and to what extent, allowing cells to respond dynamically to internal and external signals and maintain proper cellular function.

Question 8:
What are the differences between constitutive and regulated promoters, and how do they affect gene expression?

Answer: Promoters are DNA sequences located upstream of genes that initiate transcription by providing binding sites for RNA polymerase and transcription factors. They can be classified into two main types based on their activity patterns: constitutive and regulated promoters. These differences significantly influence gene expression dynamics.

Constitutive Promoters:

1. Definition:

   • Constitutive promoters drive continuous and steady transcription of their associated genes under most or all conditions.

2. Characteristics:

   • Constant Activity: Exhibit basal levels of transcription without the need for specific regulatory signals.
   • Universal Expression: Often control housekeeping genes essential for basic cellular functions, such as metabolism, cytoskeletal structure, and maintenance of cellular integrity.
   • Minimal Regulation: Less responsive to external stimuli or cellular signals compared to regulated promoters.

3. Examples:

   • CMV Promoter: Derived from the cytomegalovirus, commonly used in plasmid vectors for high-level, ubiquitous gene expression in various cell types.
   • Actin Promoter: Drives the expression of actin, a fundamental component of the cytoskeleton, in most cell types.

4. Impact on Gene Expression:

   • Stable Expression: Ensures that essential proteins are consistently produced to maintain cell viability and function.
   • Lack of Flexibility: Limited ability to modulate gene expression in response to environmental changes or developmental cues.

Regulated Promoters:

1. Definition:

   • Regulated promoters control gene expression in response to specific internal or external signals, allowing dynamic adjustment of transcription levels.

2. Characteristics:

   • Conditional Activity: Activate or repress transcription based on particular stimuli, such as hormones, stress, developmental signals, or environmental factors.
   • Tissue-Specific Expression: Often drive expression of genes required only in certain cell types or under specific physiological conditions.
   • Complex Regulation: Involve multiple transcription factors and regulatory elements (enhancers, silencers) to finely tune gene expression.

3. Examples:

   • Lac Promoter (E. coli): Regulated by the presence or absence of lactose, controlling the expression of genes involved in lactose metabolism.
   • Heat Shock Promoters: Activate in response to elevated temperatures, driving the expression of heat shock proteins that help protect the cell from stress-induced damage.
   • Steroid Hormone-Responsive Promoters: Respond to steroid hormones, regulating genes involved in processes like metabolism, development, and immune responses.

4. Impact on Gene Expression:

   • Dynamic Control: Enables cells to rapidly adjust protein synthesis in response to changing conditions, ensuring appropriate physiological responses.
   • Specificity: Allows for targeted expression of genes in specific tissues or developmental stages, contributing to cellular differentiation and specialization.
   • Flexibility: Provides the ability to upregulate or downregulate gene expression as needed, enhancing cellular adaptability and resilience.

Summary: Constitutive promoters ensure the continuous expression of essential genes, maintaining fundamental cellular functions. In contrast, regulated promoters allow for conditional and responsive gene expression, enabling cells to adapt to varying internal and external environments. The interplay between constitutive and regulated promoters is vital for the proper functioning, development, and adaptability of organisms.

Question 9:
How do non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), influence gene expression in eukaryotic cells?

Answer: Non-coding RNAs (ncRNAs) are RNA molecules that do not encode proteins but play crucial roles in regulating gene expression at various levels in eukaryotic cells. Among them, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two prominent classes with distinct mechanisms of action.

MicroRNAs (miRNAs):

1. Definition:

   • miRNAs are short, approximately 22-nucleotide-long RNA molecules involved in post-transcriptional regulation of gene expression.

2. Biogenesis:

   • Transcription: miRNA genes are transcribed by RNA Polymerase II into primary miRNAs (pri-miRNAs).
   • Processing: Drosha enzyme processes pri-miRNAs into precursor miRNAs (pre-miRNAs) in the nucleus.
   • Export and Maturation: Pre-miRNAs are exported to the cytoplasm and further processed by Dicer into mature miRNA duplexes.
   • Incorporation into RISC: One strand of the miRNA duplex is incorporated into the RNA-induced silencing complex (RISC).

3. Mechanism of Action:

   • Target Recognition: The miRNA within RISC base-pairs with complementary sequences in the 3′ untranslated regions (3′ UTRs) of target mRNAs.
   • Gene Silencing: Depending on the degree of complementarity, miRNAs can:
     • Promote mRNA Degradation: If there is near-perfect complementarity, leading to mRNA cleavage.
     • Inhibit Translation: If there is partial complementarity, blocking the translation machinery.

4. Functions:

   • Regulating Development: miRNAs control the expression of genes involved in cell differentiation and development.
   • Homeostasis: Maintain cellular balance by regulating metabolic pathways and stress responses.
   • Disease Association: Dysregulation of miRNAs is linked to various diseases, including cancer, cardiovascular disorders, and neurological conditions.

Long Non-Coding RNAs (lncRNAs):

1. Definition:

   • lncRNAs are RNA molecules longer than 200 nucleotides that do not encode proteins but have diverse roles in gene regulation.

2. Biogenesis:

   • Transcribed by RNA Polymerase II, similar to mRNAs, often with 5′ caps and poly-A tails.
   • Unlike miRNAs, lncRNAs are typically spliced and processed similarly to mRNAs but remain in the nucleus or cytoplasm without being translated.

3. Mechanism of Action:

   • Chromatin Remodeling: lncRNAs can recruit chromatin-modifying complexes to specific genomic loci, influencing the chromatin state and gene accessibility.
   • Transcriptional Regulation: Act as scaffolds or guides for transcription factors and other regulatory proteins, enhancing or repressing gene transcription.
   • Post-Transcriptional Regulation: Interact with mRNAs or other RNAs to influence splicing, stability, localization, and translation.
   • Decoys and Sponges: Bind and sequester proteins or miRNAs, preventing them from interacting with their target molecules.

4. Functions:

   • X-Chromosome Inactivation: The lncRNA XIST coats the X chromosome to initiate its inactivation in female mammals.
   • Imprinting: Regulate imprinted genes that are expressed in a parent-of-origin-specific manner.
   • Development and Differentiation: Control gene expression programs during cellular differentiation and organ development.
   • Disease Association: Aberrant lncRNA expression is implicated in cancer, metabolic disorders, and neurological diseases.

Summary: miRNAs and lncRNAs are vital regulators of gene expression in eukaryotic cells. miRNAs primarily function through post-transcriptional gene silencing by targeting mRNAs for degradation or translational inhibition. In contrast, lncRNAs exert their regulatory effects through a variety of mechanisms, including chromatin remodeling, transcriptional control, and post-transcriptional interactions. Together, these non-coding RNAs contribute to the intricate regulatory networks that maintain cellular function, development, and adaptability.

Question 10:
What are the key steps involved in the process of RNA splicing, and how does alternative splicing contribute to protein diversity?

Answer: RNA splicing is a crucial post-transcriptional process in eukaryotic cells where introns (non-coding regions) are removed from pre-mRNA, and exons (coding regions) are joined together to form mature mRNA ready for translation. Alternative splicing allows for the generation of multiple mRNA variants from a single gene, contributing significantly to protein diversity.

Key Steps in RNA Splicing:

1. Formation of the Spliceosome:

   • Spliceosome Assembly: The spliceosome, a large complex composed of small nuclear RNAs (snRNAs) and associated proteins, assembles on the pre-mRNA at specific splice sites.
   • Recognition of Splice Sites: The 5′ splice site (donor site), the branch point (typically an adenine within an intron), and the 3′ splice site (acceptor site) are recognized by the spliceosome components.

2. Catalytic Steps:

   • First Transesterification Reaction:
     • The 2′-OH of the branch point adenine attacks the 5′ splice site, creating a lariat structure by linking the intron to the branch point and releasing the upstream exon.
   • Second Transesterification Reaction:
     • The free 3′-OH of the upstream exon attacks the 3′ splice site, joining the two exons together and releasing the intron lariat for degradation.

3. Spliceosome Disassembly:

   • After splicing, the spliceosome disassembles, releasing the mature mRNA and recycling its components for future splicing events.

Alternative Splicing and Protein Diversity:

1. Definition:

   • Alternative splicing is the process by which different combinations of exons are joined together from the same pre-mRNA transcript, resulting in multiple mRNA variants from a single gene.

2. Types of Alternative Splicing:

   • Exon Skipping: Certain exons may be included or excluded from the final mRNA.
   • Mutually Exclusive Exons: Only one of two exons is included in the final mRNA.
   • Alternative 5′ Splice Site: Different 5′ splice sites are used, altering the start of an exon.
   • Alternative 3′ Splice Site: Different 3′ splice sites are used, altering the end of an exon.
   • Intron Retention: An intron is retained in the final mRNA, potentially leading to alternative protein sequences.

3. Contribution to Protein Diversity:

   • Multiple Protein Isoforms: A single gene can give rise to various protein isoforms with different functional domains, localization signals, or regulatory elements.
   • Functional Specialization: Different isoforms may perform distinct functions, respond to different signals, or be expressed in specific tissues or developmental stages.
   • Evolutionary Advantage: Increases the proteomic complexity without the need for an equivalent increase in the number of genes.

4. Regulation of Alternative Splicing:

   • Splicing Factors: Proteins that influence spliceosome assembly and splice site selection.
   • RNA-binding Proteins: Bind to specific sequences in the pre-mRNA to enhance or repress the use of certain splice sites.
   • Signaling Pathways: Cellular signals can modulate the activity of splicing factors, linking environmental or developmental cues to splicing decisions.

Examples:

• Fibronectin Gene: Undergoes alternative splicing to produce different isoforms involved in various cellular processes like adhesion, migration, and wound healing.
• Drosophila Dscam Gene: Exhibits extreme alternative splicing, generating tens of thousands of isoforms that contribute to neuronal wiring diversity.

Summary: RNA splicing, particularly alternative splicing, is a key mechanism by which eukaryotic cells achieve protein diversity. By selectively including or excluding exons and utilizing different splice sites, cells can produce multiple protein variants from a single gene, allowing for complex regulation of protein function and adaptability to diverse biological needs.

Question 11:
How does the structure of DNA facilitate its function in storing and transmitting genetic information?

Answer: The structure of DNA is intricately designed to efficiently store genetic information and ensure its accurate transmission during cell division. Several structural features contribute to these functions:

1. Double Helix Structure:

   • Description: DNA consists of two complementary strands wound around each other in a right-handed double helix.
   • Function: The double-stranded nature provides a stable structure for long-term storage of genetic information and allows for easy separation of strands during replication and transcription.

2. Nucleotide Composition:

   • Components: Each DNA nucleotide comprises a phosphate group, a deoxyribose sugar, and one of four nitrogenous bases (adenine, thymine, cytosine, guanine).
   • Base Pairing: Adenine (A) pairs with thymine (T) via two hydrogen bonds, and cytosine (C) pairs with guanine (G) via three hydrogen bonds. This complementary base pairing ensures accurate replication and transcription.

3. Antiparallel Strands:

   • Orientation: The two DNA strands run in opposite directions (one 5′ to 3′, the other 3′ to 5′).
   • Function: Antiparallel orientation is essential for the enzymes involved in replication and transcription to synthesize new strands in the correct direction.

4. Major and Minor Grooves:

   • Structure: The double helix has major and minor grooves where proteins can bind to specific DNA sequences without unwinding the helix.
   • Function: These grooves facilitate interactions with transcription factors, regulatory proteins, and other DNA-binding molecules, enabling precise regulation of gene expression.

5. Long Polymer with Minimal Replication Errors:

   • Length: DNA molecules are exceptionally long, containing the complete genetic blueprint of an organism.
   • Proofreading Mechanisms: DNA polymerases have proofreading activity that corrects errors during replication, ensuring high fidelity in the transmission of genetic information.

6. Supercoiling and Packaging:

   • Structure: DNA is further compacted into chromatin structures using histone proteins, allowing it to fit within the nucleus while maintaining accessibility for transcription and replication.
   • Function: Supercoiling helps manage the tension and spatial organization of DNA, facilitating efficient cellular processes.

7. Stability and Durability:

   • Chemical Stability: The deoxyribose sugar and the double-stranded structure confer chemical stability, protecting genetic information from degradation.
   • Durability: DNA’s structure resists hydrolysis and enzymatic degradation, ensuring the longevity of genetic information across generations.

Summary: The structural features of DNA—such as the double helix, complementary base pairing, antiparallel strands, and specific groove architecture—are fundamental to its role as the repository and carrier of genetic information. These structural attributes enable DNA to store vast amounts of information accurately, replicate efficiently with minimal errors, and interact with various proteins to regulate gene expression, ensuring the proper functioning and inheritance of genetic traits.

Question 12:
Compare and contrast the processes of transcription and translation in eukaryotic cells.

Answer: Transcription and translation are two fundamental processes in gene expression, converting genetic information from DNA into functional proteins. While they are interconnected, they involve distinct mechanisms, locations, and molecular players in eukaryotic cells.

Transcription:

1. Definition:

   • The synthesis of RNA from a DNA template, specifically producing messenger RNA (mRNA) that carries genetic information from the nucleus to the cytoplasm.

2. Location:

   • Occurs in the nucleus of eukaryotic cells.

3. Key Enzymes and Proteins:

   • RNA Polymerase II: The primary enzyme responsible for transcribing protein-coding genes.
   • Transcription Factors: Proteins that regulate the initiation and rate of transcription by binding to promoters and enhancers.

4. Stages:

   • Initiation: RNA Polymerase II, along with general and specific transcription factors, binds to the promoter region and unwinds the DNA.
   • Elongation: RNA Polymerase II synthesizes the pre-mRNA strand by adding ribonucleotides complementary to the DNA template.
   • Termination: Transcription ends when RNA Polymerase II encounters a termination signal, releasing the pre-mRNA.
   • RNA Processing: The pre-mRNA undergoes capping, splicing to remove introns, and polyadenylation to form mature mRNA.

5. Outcome:

   • Production of mature mRNA, which exits the nucleus and enters the cytoplasm for translation.

Translation:

1. Definition:

   • The synthesis of proteins by decoding the mRNA sequence into a polypeptide chain, using ribosomes and transfer RNA (tRNA).

2. Location:

   • Occurs in the cytoplasm, specifically on ribosomes attached to the endoplasmic reticulum or free in the cytosol.

3. Key Enzymes and Proteins:

   • Ribosomes: Complexes of ribosomal RNA (rRNA) and proteins that facilitate protein synthesis.
   • tRNA Molecules: Adaptors that bring specific amino acids to the ribosome based on codon-anticodon pairing.
   • Aminoacyl-tRNA Synthetases: Enzymes that attach amino acids to their corresponding tRNAs.

4. Stages:

   • Initiation: The ribosome assembles around the start codon on the mRNA, with the initiator tRNA binding to the P site.
   • Elongation: tRNAs bring amino acids to the A site, where peptide bonds are formed between amino acids, extending the polypeptide chain.
   • Termination: The ribosome encounters a stop codon, prompting the release of the completed polypeptide and disassembly of the ribosomal complex.

5. Outcome:

   • Synthesis of a functional protein based on the mRNA template.

Key Differences:

1. Function:

   • Transcription: Converts DNA information into RNA.
   • Translation: Converts RNA information into proteins.

2. Location:

   • Transcription: Nucleus.
   • Translation: Cytoplasm.

3. Molecular Machinery:

   • Transcription: Involves RNA Polymerase II, transcription factors, and RNA processing enzymes.
   • Translation: Involves ribosomes, tRNAs, aminoacyl-tRNA synthetases, and various translation factors.

4. Products:

   • Transcription: Pre-mRNA and mature mRNA.
   • Translation: Polypeptide chains that fold into functional proteins.

5. Regulation:

   • Transcription: Controlled by promoter accessibility, transcription factors, enhancers, silencers, and epigenetic modifications.
   • Translation: Regulated by mRNA stability, ribosome availability, initiation factors, and miRNA-mediated mechanisms.

Summary: Transcription and translation are sequential processes that collectively execute the central dogma of molecular biology, transforming genetic information from DNA into proteins. Transcription occurs in the nucleus, generating mRNA through the action of RNA Polymerase II and associated factors. The processed mRNA is then exported to the cytoplasm, where translation takes place on ribosomes, decoding the mRNA into a specific amino acid sequence to form proteins. Both processes are highly regulated to ensure accurate and efficient gene expression.

Conclusion: These twelve review questions cover essential aspects of DNA and RNA biology, including their structures, functions, and the processes of transcription and translation. Understanding these concepts is fundamental to grasping how genetic information is stored, processed, and utilized within eukaryotic cells. These questions and answers provide a comprehensive overview, aiding students in mastering molecular genetics and its applications in various biological contexts.