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

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