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.