chp13

Chapter 13: Transcription and RNA

Part 1: Eukaryotic Transcription and RNA Processing

The Central Dogma of Molecular Biology

  • Definition: The Central Dogma describes the flow of genetic information within a biological system.
      - Directed by Francis Crick.

  • Typical Order of Information Flow:
      - DNA → RNA → Protein (Fig 10.17a)
      - Exceptions: Some exceptions to this flow are recognized (Fig 10.17b).

An Early RNA World

  • Role of RNA in early life forms: RNA is thought to have played a critical role in the emergence of early life.

  • Relationship between DNA, RNA, and Protein:
      - DNA ?
      - RNA ?
      - Protein

  • Functions of RNA:
      - Catalytic RNAs (ribozymes) facilitate various biochemical reactions including:
        - Cleaving bonds and rearranging molecular structures
        - Catalyzing peptide bond formation by ribosomes
        - Removing introns from precursor RNAs
        - Antiviral activity (e.g., CRISPR systems)
        - RNA interference mechanisms
        - Storage of genetic information

Fundamentals of RNA

  • Chemical Composition:
      - Contains ribose sugar and uracil instead of thymine as a pyrimidine base.

  • Structure of RNA:
      - Primary Structure: Linear sequence of nucleotides.
      - Secondary Structure: Involves regions of complementarity and molecular folding.

Comparison of DNA and RNA Structures (Table 13.1):

  • Characteristics:
      | Characteristic | DNA | RNA |
      |-------------------------------------|--------------------|--------------------|
      | Composed of nucleotides? | Yes | Yes |
      | Type of sugar? | Deoxyribose | Ribose |
      | Presence of 2'–OH group? | No | Yes |
      | Bases | A, G, C, T | A, G, C, U |
      | Nucleotides joined by | phosphodiester bonds | phosphodiester bonds |
      | Double or single stranded? | Usually double | Usually single |
      | Stability | Stable | Less stable than DNA |

Classes of Cellular RNA

  • RNA molecules serve various functions and can be found in different cellular locations. (Table 13.2)
      - Ribosomal RNA (rRNA):
        - Location: Cytoplasm in prokaryotes and eukaryotes
        - Function: Structural and functional components of the ribosome.
      - Messenger RNA (mRNA):
        - Location: Nucleus and cytoplasm in prokaryotes and eukaryotes
        - Function: Carries genetic code for proteins.
      - Transfer RNA (tRNA):
        - Location: Cytoplasm in prokaryotes and eukaryotes
        - Function: Assists in incorporating amino acids into polypeptide chains.
      - Small nuclear RNA (snRNA):
        - Location: Nucleus in eukaryotes
        - Function: Processing of pre-mRNA.
      - Small nucleolar RNA (snoRNA):
        - Location: Nucleus in eukaryotes
        - Function: Processing and assembly of rRNA.
      - MicroRNA (miRNA):
        - Location: Nucleus and cytoplasm in eukaryotes
        - Function: Inhibits translation of mRNA.
      - Small interfering RNA (siRNA):
        - Location: Nucleus and cytoplasm in eukaryotes
        - Function: Triggers degradation of other RNA molecules.
      - Piwi-interacting RNA (piRNA):
        - Location: Nucleus and cytoplasm in eukaryotes
        - Function: Suppress transcription of transposable elements in reproductive cells.
      - CRISPR RNA (crRNA):
        - Location: Prokaryotic cells
        - Function: Assists in destruction of foreign DNA.
      - Long noncoding RNA (lncRNA):
        - Location: Nucleus and cytoplasm in eukaryotes
        - Function: Involved in a variety of cellular functions.
      - Note: All eukaryotic RNAs are synthesized in the nucleus.

Transcription of an RNA Molecule from a DNA Template

  • Definition: The process of synthesizing RNA from a DNA template is known as transcription.

  • Three Major Requirements for Transcription:
      1. DNA Template: Only one strand of the gene’s DNA is used as a template.
      2. Ribonucleotide Triphosphates (rNTPs): Building blocks for RNA synthesis.
      3. RNA Polymerase Protein Complex: Recognizes genes and synthesizes RNA.

DNA Strand Orientation and Transcription

  • Strands of DNA: Genomic DNA is double-stranded; only one strand is used for transcription, known as the template strand.
      - Transcribed Strand (Template Strand): Used for synthesizing RNA.
      - Nontemplate Strand: Complementary to the template and reads the same as the RNA with the exception of T → U.

  • Visualization: Transcription can be visualized for some genes in specific cells (Fig 13.3).

Directions of Transcription

  • Transcription Directionality: Genes are directional, transcribed in only one direction to produce a specific product.
      - 5' to 3' Synthesis: RNA synthesis occurs in the 5' to 3' direction, following base complementarity and hydrogen bonding, while maintaining antiparallel orientation between DNA and RNA strands during synthesis.

Gene Structure and Transcription Units

  • Gene Components: Genes consist of several sequence regions, including:
      - Promoter Sequence Region: Located upstream of the transcription start site.
      - RNA Coding Region: Contains the actual information coding for the RNA.
      - Terminator: Signals the end of transcription.

  • Promoter Function:
      - Not typically transcribed into RNA (except eukaryotic tRNA genes).
      - Orient the RNA polymerase complex at the gene start and help regulate transcription timing, location, and amount.

Requirements for Transcription in Bacteria

  • Bacterial RNA Polymerase (RNA Pol):
      - Multisubunit complex comprising core polymerase subunits:
        - α, α, β, β’, ω (core components) and σ (sigma) factor which combines with core to form the holoenzyme.
      - Ribonucleotides (NTPs) for RNA synthesis: A, G, U, C (U substitutes T from DNA).
      - No primer is necessary to initiate synthesis.

Phases of Bacterial Transcription

  • Phases Described:
      1. Initiation:
        - RNA polymerase and associated proteins bind to the promoter to begin transcription and join initial nucleotides.
      2. Elongation:
        - RNA polymerase moves along the template, elongating the RNA molecule in the 5’ to 3’ direction.
      3. Termination:
        - RNA polymerase disengages from the template upon reaching the end of the RNA coding sequence.

Initiation Details

  • Bacterial Promoters:
      - Share common recognizable features.
      - -10 Consensus (Pribnow Box):
        - Sequence: 5’ TATAAT 3’
        - Location: 10 base pairs upstream from the transcription start site.
      - -35 Consensus Sequence:
        - Sequence: 5’ TTGACA 3’
        - Location: Approximately 35 base pairs upstream from the transcription start site.

  • Consensus Sequences:
      - Compilation of the most frequently encountered nucleotides at each site across similar promoter sequences.

Stabilization of RNA Polymerase

  • Holoenzyme Action:
      - Binding to common consensus sequences stabilizes RNA polymerase.

  • Transcription Bubble:
      - Opening occurs due to tension and energy changes in DNA supercoiling.

Starting RNA Synthesis

  • Initiation Phases:
      - Formation of a transcription bubble allows RNA polymerase to begin linking two rNTPs.
      - Abortive Initiation: Early linked nucleotides sometimes do not result in continued synthesis.
      - Promoter Escape: Transition to effective elongation once several successful nucleotides are linked.
      - Decoupling of sigma factor typically occurs soon after this phase.

Elongation of Bacterial Transcription

  • Continued Synthesis:
      - Conducted by core RNA polymerase until a terminator is reached.
      - DNA-RNA Hybrid: Only a short region (8 bp) is maintained in the transcription bubble.
      - Average addition of nucleotides: 30-50 nucleotides per second, with variations in speed due to pausing.
      - Topoisomerases Management: Supercoiling energy remains balanced during transcription.
      - Some proofreading occurs but less stringent compared to DNA replication fidelity.

Termination of Bacterial Transcription

  • Mechanisms for Termination:
      - Rho-Dependent Termination:
        - The (rho) protein binds to a rut site in the RNA transcript, dislodging RNA polymerase from the DNA template.
      - Rho-Independent Termination:
        - Formation of a hairpin structure in the RNA sequence at inverted repeat sequences followed by a stretch of uracils causes dissociation due to weak hydrogen bonds.

Conclusion: Mechanisms of Transcription

  • Final notes include specific errors in illustrations of transcription.

  • Correct identification of promoter regions and the proper sequence of RNA synthesis initiation is fundamental for understanding transcription.

  • A continual interim review of RNA-synthesizing processes helps clarify molecular biology's mechanisms.

Figures and Tables

  • Refer to Figures 10.17a, 10.17b, 13.2, 13.3, 13.5, and illustrative diagrams showing RNA synthesis processes.

  • Key tables include Table 13.1 and Table 13.2.