Transcription in Prokaryotes

• Transcription is the process of copying the information in a gene from DNA to RNA, always occurring on only one of the DNA strands

  
• RNA polymerases, enzymes that synthesize RNA from a DNA template, copy the template DNA strand in the 3' to 5' direction and synthesize the RNA in the 5' to 3' direction
• Order of events in Transcription:

  1. Binding of polymerases to the initiation site, the promoter
  2. Unwinding of the DNA double helix by a helicase
  3. Synthesis of RNA based on the sequence of the DNA template strand using nucleoside triphosphates (NTPs)
  4. Termination of synthesis
• Prokaryotic RNA Polymerase:

  • Core Enzyme: synthesizes RNA but initiation point is non-specific
  • Holoenzyme: includes a sigma factor required to initiate RNA synthesis at specific locations in the DNA, termed the promoter
• Promoters:

  • Promoter regions in DNA where RNA polymerase binds to initiate transcription
  • Consensus sequences are "best average" sequences found in promoters, with mutations affecting promoter functionality
• Termination of Transcription in Prokaryotes:

  • Two major mechanisms: Rho-independent (intrinsic) and Rho-dependent
  • Rho-independent termination involves a stem-loop structure in the RNA transcript
  • Rho-dependent termination involves the Rho protein binding to RNA and moving along it to cause termination
• Not all RNA is translated into protein; some are:

  • structural (ribosomal RNA)
  • functional (transfer RNA)
  • chromosomal (some viruses)
• RNA Polymerase is formerly called DNA - dependent RNA polymerases.
• Properties of RNA Polymerases:

  1. template dependent, requires double stranded DNA
  2. require the four nucleoside triphosphates (ATP, GTP, CTP, UTP)
  3. copy (read) the template DNA strand in 3' to 5' direction
  4. synthesize RNA in 5' to 3' direction
• The STOP codon in the genetic code for the end of peptide synthesis is NOT the end of termination in RNA synthesis.
• Prokaryotic RNA Polymerase: Core Enzyme

  A) alpha

  B) beta

  C) beta'

  
• Prokaryotic RNA Polymerase: Holoenzyme

  A) sigma

  
• David Pribnow - compared promoter regions of five genes from E. coli, found conserved sequences or Pribnow box.
• Promoters

  • For any given gene, RNA synthesis always starts at the same point on the DNA, the promoter. What is a promoter?


  Hypothesis: Because one RNA polymerase copies every gene and binds to the promoter in each gene to do so, the promoters in different genes must have similarities. Similarities in DNA must lie in the sequence of nucleotides so the promoters of every gene must have the same sequence of nucleotides.
• The Pribnow box lies 10 nucleotides from the transcription start point (TSP). A second was later found 80 nucleotides away.

  A) Pribnow box

  
• Consensus sequences

  • few genes contain a sequence that is only a few nucleotides different.
  • “best average”

  
• Initiation

  1. Sigma binds to promoter region, recognizing both the -35 and -10 regions. The resulting structure is termed a closed promoter complex. The promoter is rich in A and T.
  2. After the DNA strands have been separated at the promoter region by the helicase activity of the sigma subunit, forming an open promoter complex. The core subunit (aabb') can then start to synthesize RNA.
  3. Following initiation, the sigma subunit is released after approx. 10 ribonucleotides have been polymerized.
• Elongation

  • Synthesis of the RNA strand continues until the core polymerase reaches the termination site.
• The Rho-independent termination signal is a stretch of 30-40 bp sequence, consisting of many GC residues followed by a series of T ("U" in the transcribed RNA). As transcription proceeds, the two GC rich regions base pair. This leaves a short poly U rich region, which cannot pair strongly enough to hold the RNA onto the DNA. The polymerase comes off with it.
• Rho-independent termination

  A) stem loop

  
• Rho-dependent termination

  • In vitro, E. coli RNA polymerase holoenzyme transcribes DNA into a very long RNA; natural length RNA is restored by the addition of a protein factor, called rho (r).
  • Analysis of termination sites dependent on rho revel a stem loop structure near the 3‘ end of the RNA, with NO U-rich tale.
  • Rho binds to RNA and can, if provided with ATP, move along the RNA.
  • Rho also has ATP-dependent helicase activity

  
• Model for rho termination

  • It has been established that six Rho proteins form a hexamer to terminate transcription, but the precise mechanism is not clear.

  1. The Rho hexamer first binds to the RNA transcript at an upstream site which is 70-80 nucleotides long and rich in C residues
  2. Upon binding, the Rho hexamer moves along the RNA in the 5'-3' direction, trying to catch up with the RNA polymerase.
  3. When the polymerase pauses, which happens when secondary structures form near the 3 end of the RNA, rho catches up and melts the RNA-DNA duplex in the replication bubble, causing termination.
• Model for rho termination

  A) rho

  
• Termination of transcription can serve a role in regulating gene expression in prokaryotes