TR

Transcription

Introduction

  • Gene: an organized unit of DNA sequences that enables a segment of DNA to be transcribed into RNA and ultimately results in the formation of a functional product
    • Other genes code for RNA itself as a product
    • Transfer RNA (tRNA): translates mRNA into amino acids
    • Ribosomal RNA (rRNA): part of ribosomes
  • Eukaryotes also have an intervening step called RNA processing
    • RNA processing: pre-mRNA is processed into active mRNA
  • Some genes do not encode polypeptides
    • An RNA is the final functional product
    • Structural RNAs
    • Regulatory RNAs

ncRNA Functions

  • Scaffold: bind to multiple components such as proteins, act as scaffold for formation of a complex
  • Guide: guide one molecule to a specific location in the cell
  • Alteration of protein function or stability: binds to a protein, can affect ability of the protein to be a catalyst, ability of the protein to bind other molecules, protein stability
  • Ribozymes: RNA molecule with catalytic function
  • Blocker: prevents or blocks a cellular process from happening
  • Decoy: recognize other ncRNAs and sequester them, preventing them from working

Differences in Prokaryotic and Eukaryotic Transcription

  • Prokaryotes
    • All RNA species are synthesized by a single RNA polymerase
    • mRNA is translated during transcription
    • Genes are contiguous segments of DNA that are colinear with the mRNA that is translated into a protein.
    • mRNAs are often polycistronic
  • Eukaryotes
    • 3 different RNA polymerases for the different classes of RNA molecules.
    • mRNA is processed before transport to the cytoplasm. Caps and tails are added and introns are removed.
    • Genes are often split. Exons are separated by introns
    • mRNAs are monocistronic

RNA Polymerase (RNAP)

  • Synthesizes RNA in a 5’🡪 3’ direction

  • Requires a DNA template (read in a 3’ to 5’ direction)

  • Are large complexes of proteins

  • Prokaryotic: single type of RNAP

    • Contains four catalytic subunits and a single regulatory subunit known as sigma (s).
    • α2ββ’
    • Distinct sigma factors
    • Are sequence specific DNA binding proteins
    • Reduce non-specific DNA binding of the RNAP
    • Increase specific binding to the promoters

  • Eukaryotic RNA polymerases have different roles in transcription

    • Polymerase I: makes a large precursor to the major rRNA (5.8S,18S and 28S rRNA in vertebrates)
    • Polymerase II: synthesizes hnRNAs, which are precursors to mRNAs. It also make most small nuclear RNAs (snRNAs)
    • Polymerase III: makes the precursor to 5SrRNA, the tRNAs and several other small cellular

    and viral RNAs.

Promoters

  • Promote assembly of the RNA pre-initiation complex
  • Position the transcription start site
  • Control the directionality of transcription
  • Location and orientation of promoter dictates which strand of dna is used for transcription

Directions of Transcription

  • Direction of transcription and which DNA strand used varies among genes
  • In all cases, synthesis of RNA transcript is 5’ to 3’ and DNA template strand reads 3’ to 5’
  • Antisense strand: used a a template
  • Sense strand: not the template strand and has the same sequence as the RNA molecule

Promoter Sequences

  • Prokaryotes
    • A sequence of TATAAT centered 10 nucleotides upstream of the +1 site and is called the Pribnow box
    • The sequence TTGACA centered at position -35 and is the site of sigma factor binding
  • Eukaryotes
    • TATA box located about 25 nucleotides upstream from the transcriptional start site.
    • CAAT box at position  -70

Eukaryotic Promoters

  • The basal or core promoter is found in all protein-coding genes.
  • Many different genes and many different types of cells share the same transcription factors
    • Not only those that bind at the basal promoter but even some of those that bind upstream
  • What turns on a particular gene in a particular cell is probably the unique combination of promoter sites and the transcription factors that are chosen

Three Stages of Transcription

  1. Initiation
    • Recognition step
    • Promoter required
    • Stage completed when DNA strands separate near promoter to form open complex
  2. Elongation
    • RNA polymerase synthesizes RNA
    • Template or coding strand used for RNA synthesis
      • Noncoding strand is not used
    • Synthesized 5’ to 3’
  3. Termination
    • RNA polymerase reaches termination sequence
    • Causes both the polymerase and newly-made RNA transcript to dissociate from DNA

Inducible Genes

  • Inducible genes: genes whose expression is turned on by the presence of some substance
    • Lactose induces expression of the lac genes
    • An antibiotic induces the expression of a resistance gene
  • Catabolic pathways

Repressible Genes

  • Repressible genes: genes whose expression is turned off by the presence of some substance (co-repressor)
    • Tryptophan represses the trp genes
  • Biosynthetic pathways
    • Co-repressor is typically the end product of the pathway

Combinatorial Control

  1. Activators: activator proteins stimulate RNA polymerase to initiate transcription
  2. Repressors: repressor proteins inhibit RNA polymerase from initiating transcription
  3. Modulation: small effector molecules, protein–protein interactions, and covalent modifications can modulate activators and repressors
  4. Chromatin: activator proteins promote loosening up of the region in the chromosome where a gene is located, making it easier for RNA polymerase to transcribe the gene
  5. DNA Methylation: usually inhibits transcription, either by blocking an activator protein or by recruiting proteins that make DNA more compact