8-2

Week Eight Part Two: Transcription Overview

  • Introduction

    • Focus on transcription process.

    • Comparison to DNA replication.

    • Depiction of chemical reactions involved in transcription - reactants on the left and products on the right.

Transcription Process

  • Template Requirement

    • A template strand is essential for transcription.

    • Template strand runs in the 3' to 5' direction.

    • The complementary strand (non-template) runs in the 5' to 3' direction.

    • RNA polymerase synthesizes RNA in the 5' to 3' direction, similar to DNA polymerase.

  • Necessary Components

    • Template strand (3' to 5').

    • Ribonucleotide triphosphates (rNTPs) - RNA's building blocks.

      • Four rNTPs needed: Adenosine (A), Cytidine (C), Guanosine (G), Uridine (U).

      • Importance of triphosphates with sugar ribose.

    • Formation of the sugar-phosphate backbone.

      • Pyrophosphate is released during nucleotide addition, leading to phosphodiester bond creation.

Key Differences from DNA Replication

  • No Primer Required

    • RNA polymerase initiates RNA synthesis de novo (without a primer).

  • Nucleotide Differences

    • Uracil incorporated instead of thymine.

    • rNTPs are used instead of deoxynucleotides (dNTPs).

  • Strand Usage

    • Only one strand is selected as the template (3' to 5').

      • Known as the DNA antisense strand.

    • Complementary RNA strand synthesized runs 5' to 3'.

    • The other strand is called the DNA sense strand.

RNA Polymerase Functionality

  • Directionality and Functioning

    • RNA polymerase works 5' to 3' directionally.

    • Lacks proofreading capability - unlike DNA polymerase which can proofread.

    • Functions to unwind DNA strands, unlike DNA polymerase that requires helicase.

  • Stages of Transcription

    • Initiation

    • Elongation

    • Termination

Prokaryotic Gene Structure

  • Overview

    • Consists of two DNA strands: Template 3' to 5' and non-template strand.

    • Key regions include the promoter, transcription start site, coding region, and termination site.

  • Importance of Promoter

    • Directs RNA polymerase to the correct direction for synthesis.

    • Initiation site is crucial for the start of transcription.

Distinction Between RNA and DNA Polymerase

  • Workflow Direction

    • Both operate uni-directionally (5' to 3').

    • DNA polymerase can backtrack for proofreading; RNA polymerase cannot.

  • Mechanism of Action

    • RNA polymerase unwinds the DNA to synthesize RNA and forms a transcription bubble.

Prokaryotic Transcription Detailed

  • Holoenzyme Formation

    • Composed of RNA polymerase and sigma factor.

    • Essential for initiating transcription in prokaryotes.

  • Function of Sigma Factor

    • Helps RNA polymerase bind to unique promoter sequences.

    • Antibiotics can target sigma factor to inhibit bacterial transcription (e.g., against tuberculosis).

  • Components of Holoenzyme

    • Alpha subunit: Assembly of holoenzyme core.

    • Beta subunit: Binding site for rNTPs.

    • Beta prime: DNA template binding region.

    • Omega: Stabilizes holoenzyme structure.

    • Sigma: Crucial for initiation of transcription.

Promoter Elements in Prokaryotes

  • Key Sequencing Elements

    • Consists of -10 and -35 sequences necessary for sigma binding.

    • -10: Known as TATA box (TATAAT).

    • TATA box is important for RNA polymerase positioning.

  • Initiation Site

    • Transcription start site (denoted as +1).

    • TATAAT box located around -10; some variation allowed.

Eukaryotic Transcription Overview

  • Promoter Structures

    • Contains TATA box located around the -25 position.

    • Eukaryotic promoters differ from prokaryotic ones with more complex recognition elements.

Steps of Transcription

  • Initiation

    • Requires the presence of a promoter.

    • RNA polymerase unwinds strands; helicase activity inherent in RNA polymerase.

    • Promotion orientation is essential in determining transcription direction.

  • Elongation

    • Sigma factor is released after initiation.

    • New nucleotides joined to the growing RNA strand at the 3' end.

    • RNA polymerase continues to unwind and synthesize RNA from 5' to 3'.

  • Termination

    • Termination occurs when RNA polymerase reaches the terminator sequence.

    • RNA transcript is released along with RNA polymerase.

    • Types of terminators: Row-dependent and row-independent terminators.

  • Conclusion of Terminators

    • Row-dependent: Requires a specific protein factor (row factor).

    • Row-independent: Functions without additional factors for termination of transcription.