02-13-2025 Lecture Outline 3

Page 1: Introduction to Transcription

  • Overview of the process of converting DNA to RNA.

Page 2: Types of RNA Produced by Transcription

  • Transcription involves copying a short region of DNA into RNA across all organisms.

    • Types of RNA:

      • mRNA or pre-mRNA

      • rRNA

      • tRNA

  • Specific to organisms:

    • Archaea and Eukaryotes:

      • snRNA, snoRNA

      • siRNA, miRNA, piRNA, lncRNA

    • Bacteria and Archaea:

      • crRNA (CRISPR—clustered regularly interspaced short palindromic repeats)

  • Gene Definition: Copied portion of DNA.

  • RNA Properties:

    • Contains ribose, uracil, and is single-stranded, allowing tertiary structure formation.

Page 3: RNA Tertiary Structure

  • RNA can fold into complex tertiary structures that are critical for function.

Page 4: Prokaryotic Transcription Overview

  • Introduction to the transcription mechanism in prokaryotic organisms.

Page 5: Initiation of Prokaryotic Transcription

  • σ Factor Role:

    • Binds to RNA polymerase and slides along DNA to find promoter.

    • Binds to promoter and causes double helix opening.

    • Dissociates, allowing elongation to occur.

Page 6: Prokaryotic RNA Polymerase Structure

  • After σ factor dissociation:

    • A jaw-like structure forms, holding DNA in place.

    • rNTP uptake and RNA exit channels are formed.

    • Rudder-like protrusion pries apart the DNA-RNA double helix, allowing single strands to re-anneal.

Page 7: Termination of Transcription in Prokaryotes

  • Polarity termination sequence is reached:

    • A special RNA sequence gets transcribed, folding into a hairpin structure, forcing the jaw to open and releasing the transcript.

    • RNA polymerase dissociates from the DNA.

Page 8: Eukaryotic Transcription Overview

  • Introduction to eukaryotic transcription with a focus on complexity and variety of polymerases.

Page 9: Eukaryotic RNA Polymerases

  • There are three eukaryotic polymerases:

    • POL I: Transcribes rRNA.

    • POL II: Transcribes pre-mRNA.

    • POL III: Transcribes tRNA and other small RNAs.

  • Functionally similar to bacterial polymerase.

Page 10: Initiation in Eukaryotes

  • Involves general transcription factors (TFs).

    • Multi-subunit protein family.

    • Some TFs bind to the double helix before polymerase binding, others bind to polymerase first.

Page 11: Details of POL II Initiation (mRNA)

  • Promoter sequence located ~25 bp upstream of transcription start; typically involves a TATA box.

  • TFIID binds to the promoter and distorts the double helix, which attracts other transcription factors to initiate transcription complex formation.

Page 12: TBP-DNA Binding and Transcription Initiation Complex Formation

  • Sequence of steps involved in TBP and associated factors binding is detailed.

  • Involves assembling a complex of various transcription factors with RNA polymerase II.

Page 13: POL II Initiation Mechanism

  • TFIIH Role:

    • Contains DNA helicase, accessing the template strand.

    • Synthesizes a short pre-mRNA strand.

    • Phosphorylates POL II's C-terminus, transitioning it out of the initiation phase.

Page 14: Eukaryotic Initiation Summary

  • The depicted steps summarize the initiation process, including the involvement of multiple transcription factors.

Page 15: POL II CTD Phosphorylation

  • Detailed structure of the C-terminal domain (CTD) of RNA Polymerase II showing heptapeptide repeats.

  • Role of phosphorylation in regulating transcription initiation and elongation.

Page 16: Additional Initiation Proteins in Eukaryotic Transcription

  • Activators, repressors, mediators, and chromatin-remodeling enzymes are part of the initiation control.

Page 17: mRNA Processing

  • Bacterial mRNA is directly translated, while eukaryotic pre-mRNA undergoes significant modifications.

  • Key Modifications Include:

    • Capping of the 5' end.

    • Splicing: Removing introns and ligating exons.

    • Polyadenylation of the 3' end.

Page 18: Coupling Processing with Elongation

  • POL II’s C-terminal phosphorylation couples transcription and mRNA processing, facilitating the binding of processing proteins to emerging mRNA.

Page 19: Pre-mRNA Processing Proteins

  • Various proteins associated with the processing of pre-mRNA are highlighted, including those responsible for capping, splicing, and polyadenylation.

Page 20: The 5’ “Cap” of mRNA

  • Added by a complex of enzymes, serves to mark the mRNA for nuclear export and plays a role in translation regulation.

Page 21-24: Steps of 5' Cap Addition

  • Three Steps to 5’ Cap Addition:

    1. Phosphatase removes a phosphate from the 5' end.

    2. Guanyl transferase adds GMP to the 5' end.

    3. Methyl transferase adds a methyl group to the guanosine.

Page 25: Pre-mRNA Splicing Overview

  • Splicing removes introns while retaining exons; alternative splicing allows different protein forms to be produced from a single gene.

Page 26: Example of Alternative Splicing

  • The a-tropomyosin gene serves as an example of alternative splicing resulting in varied mRNA for different tissues.

Page 27-28: The Spliceosome Mechanism

  • The spliceosome includes a core of snRNA-protein complexes that undergo dynamic rearrangements using ATP hydrolysis to facilitate splicing reactions.

Page 29: The Splicing Reaction Steps

  • Detailed steps of intron removal and exon ligation in the splicing process are described, including the formation of a lariat structure.

Page 30: RNA-RNA Rearrangements in Splicing

  • Importance of ensuring that splice sites are correctly identified through RNA-RNA interactions.

Page 31: Polyadenylation Process

  • Discussion of how poly-A signals lead to cleavage and the addition of poly-A tails to mRNA for stability and regulation.

Page 32: RNA Types and Gene Expression

  • Only a few mRNA copies are needed for protein production, while non-coding RNAs need many copies, often encoded across multiple chromosomes.

Page 33: Ribosomal RNA (rRNA)

  • Synthesized by POL I, characterized by modifications such as methylation and isomerization for proper function.

Page 34: Nucleolus Functions

  • The nucleolus is a key site within the eukaryotic nucleus where ribonucleoprotein assembly occurs.

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