INTRODUCTION TO RNA AND TRANSCRIPTION

  • Overview of RNA, its structure, and function in the process of transcription.

RNA STRUCTURE

  • RNA is usually single-stranded.
  • Contains ribose sugar and has uracil (U) instead of thymine (T) found in DNA.
  • Primary Structure Example:
    • Sequence: 5' AUGCGGCUACGUAACGAGCUUAGCGCGUAUACCGAAAGGGUAGAAC 3'
    • Note on Ribose: Has a hydroxyl (OH) group at the 2'-carbon whereas DNA has hydrogen (H).
    • Implication: RNA is more reactive than DNA.
    • Secondary Structure: RNA can fold due to hydrogen bonding between complementary bases on the same strand, leading to complex structures.

TRANSCRIPTION OVERVIEW

  • Definition: Transcription is the process of reading a DNA strand to create RNA.
  • Central Dogma of Molecular Biology:
    • DNA ⇌ RNA (Transcription) ⇌ Protein (Translation)
    • Includes concepts of replication and reverse transcription.

TEMPLATE AND NON-TEMPLATE STRAND

  • Template (coding) vs. Non-template (strand):
    • RNA synthesis occurs complementary and antiparallel to the template strand.
    • RNA polymerase reads the template strand in the 3' to 5' direction.
    • mRNA is assembled from nucleotides in the 5' to 3' direction.
    • Complementarity: Non-template is similar to mRNA, only differing by T in DNA replaced with U in RNA.

TRANSCRIPTION UNIT

  • Definition: A transcription unit comprises a sequence of nucleotides in DNA that encodes a single RNA molecule.
  • Structure: Typically includes:
    • Promoter: where transcription begins
    • RNA-coding region: sequence coding for RNA
    • Terminator: sequence marking the end of transcription.

STAGES OF TRANSCRIPTION

  • Transcription occurs in three stages:
    1. Initiation: Assembly of transcription apparatus at the promoter and beginning RNA synthesis.
    2. Elongation: Involves unwinding the DNA and adding nucleotides to the RNA strand.
    3. Termination: Recognition of the end of the transcription unit and separating RNA from DNA.

INITIATION

  • Process includes:
    • Promoter recognition by RNA polymerase.
    • Formation of a transcription bubble.
    • Creation of initial rNTP bonds and escaping the promoter.

CONSENSUS SEQUENCES

  • Consensus Sequence: Most common nucleotides at specific DNA locations, e.g., TATA box 5'-TATAAA-3'.
    • Used to identify promoter regions across genes.

BACTERIAL PROMOTER CONFIGURATION

  • Consensus sequences in bacterial promoters:
    • Common elements at positions -35 (TTGACA) and -10 (TATAAT).

RNA POLYMERASE IN BACTERIA

  • Types of RNA polymerases in bacteria:
    • Core enzyme: Set of five subunits involved in elongation.
    • Sigma factor: Assists in locating promoters.
    • Holoenzyme: Complete enzyme complex for transcription.

INITIATION IN BACTERIA

  1. Sigma factor associates with core enzyme to form a holoenzyme.
  2. Holoenzyme binds to promoter at -35 and -10 regions.
  3. Tightly binds, unwinding the DNA.
  4. Sigma factor released as RNA polymerase advances.

ELONGATION OF TRANSCRIPTION

  • Characteristics during elongation:
    • RNA polymerase creates a transcription bubble of about 18 nucleotides.
    • Integrity of DNA is restored upstream (rewinds).
    • If a mismatch occurs during transcription, the polymerase can backtrack and remove the faulty nucleotide.

TERMINATION OF TRANSCRIPTION IN BACTERIA

  • Rho-Dependent Termination:
    • Rho factor binds to RNA and approaches the RNA polymerase to help terminate transcription upon reaching a terminator sequence.
  • Rho-Independent Termination:
    • Involves inverted repeats followed by a string of adenines, causing RNA polymerase to pause leading to dissociation.

EUKARYOTIC TRANSCRIPTION OVERVIEW

  • More complex than bacterial transcription, utilizing multiple RNA polymerases:
    • RNA Polymerase I: Transcribes large rRNAs.
    • RNA Polymerase II: Transcribes pre-mRNA and various small RNAs.
    • RNA Polymerase III: Transcribes tRNA and small rRNAs.

EUKARYOTIC PROMOTERS

  • Complexity in eukaryotic promoters includes:
    • Regulatory sequences such as TATA boxes, BRE, and others.
    • Involvement of transcription factors for recognition and binding.

ELONGATION IN EUKARYOTES

  • RNA polymerase maintains a transcription bubble and incorporates nucleotides matching the DNA template.

TERMINATION IN EUKARYOTES

  • Varies by polymerase:
    • Polymerase I requires termination factors similar to rho.
    • Polymerase II uses protein Rat1 to help degrade RNA trailing after cleavage near 3' end.
    • Polymerase III stops after encountering terminator sequences that include uracil strings.

SUMMARY OF TRANSCRIPTION

  • RNA is generally single-stranded and plays various roles in transcription.
  • Initiation is mediated by promoter sequences and other proteins.
  • RNA polymerases synthesize RNA from the template strand and resemble the non-template strand, except for uracil and thymine differences.
  • Diverse mechanisms exist for transcription termination.