Prokaryotic transcription is a vital process where DNA is converted into RNA. It involves three key steps: initiation, elongation, and termination. Each step plays a crucial role in accurately producing RNA molecules that guide protein synthesis in bacterial cells.

Promoters and termination mechanisms are essential for regulating gene expression in prokaryotes. Promoters control when and how often genes are transcribed, while termination signals ensure transcription stops at the right place. These elements allow bacteria to adapt quickly to changing environments.

Prokaryotic Transcription

Steps of prokaryotic transcription

1. Initiation

   • RNA polymerase binds to the promoter region located upstream of the gene to be transcribed (lac operon)
   • Promoter contains specific -10 and -35 consensus sequences that are recognized by the RNA polymerase (TATAAT and TTGACA)
   • Sigma factor, a subunit of RNA polymerase, helps the enzyme recognize and bind to the promoter sequence
   • Upon binding, the DNA double helix unwinds and separates, forming a transcription bubble that allows access to the template strand

2. Elongation

   • RNA polymerase reads the template strand in the 3' to 5' direction, synthesizing the complementary RNA strand
   • Ribonucleotides (ATP, UTP, CTP, GTP) are added to the growing RNA chain in the 5' to 3' direction, following the base-pairing rules
   • RNA polymerase catalyzes the formation of phosphodiester bonds between the ribonucleotides, extending the RNA strand
   • Sigma factor is released from the RNA polymerase complex after transcription initiation, allowing the enzyme to proceed with elongation
   • RNA polymerase continues to synthesize the RNA molecule until it reaches a specific termination signal on the DNA template

3. Termination

   • Transcription termination in prokaryotes occurs through two main mechanisms: Rho-dependent and Rho-independent termination
   • Rho-dependent termination involves the Rho protein, an ATP-dependent RNA helicase that binds to the nascent RNA and causes the RNA polymerase to dissociate from the DNA template (trp operon)
   • Rho-independent termination, also known as intrinsic termination, occurs when the nascent RNA forms a stable hairpin loop structure followed by a string of uracil residues, causing the RNA polymerase to stall and dissociate from the DNA template (his operon)

Promoters in prokaryotic gene regulation

• Promoters are DNA sequences located upstream of the transcription start site that play a crucial role in regulating gene expression in prokaryotes (lac promoter)
• RNA polymerase recognizes and binds to the promoter region to initiate the transcription process
• The strength of a promoter determines the frequency at which transcription initiation occurs
  • Strong promoters have consensus sequences that closely match the ideal -10 (TATAAT) and -35 (TTGACA) sequences, leading to higher rates of transcription
  • Weak promoters have sequences that deviate from the consensus, resulting in lower transcription rates and reduced gene expression
• Regulatory proteins can bind to specific sites within or near the promoter region and affect the transcription process
  • Activator proteins enhance transcription by recruiting RNA polymerase to the promoter or facilitating its binding (CAP protein)
  • Repressor proteins inhibit transcription by blocking RNA polymerase from binding to the promoter or preventing its progression along the DNA template (lac repressor)
• Environmental signals, such as changes in nutrient availability (glucose), temperature, or other factors, can influence the binding of regulatory proteins to the promoter, allowing bacteria to adapt to changing conditions
• Transcription factors, including activators and repressors, play a crucial role in modulating gene expression by interacting with the promoter region

Prokaryotic transcription termination mechanisms

• Rho-dependent termination
  • Requires the involvement of the Rho protein, an ATP-dependent RNA helicase
  • Rho binds to specific sites on the nascent RNA called Rho utilization sites (rut) and moves along the RNA in the 5' to 3' direction
  • Rho uses energy from ATP hydrolysis to translocate along the RNA and catch up to the RNA polymerase
  • When Rho reaches the RNA polymerase, it causes the enzyme to dissociate from the DNA template, effectively terminating transcription
• Rho-independent termination
  • Also known as intrinsic termination, this mechanism relies on the formation of a specific secondary structure in the nascent RNA
  • The termination signal consists of a stable hairpin loop structure followed by a string of uracil residues
  • RNA polymerase stalls at the uracil-rich region due to the weak base-pairing between the RNA and DNA strands
  • The stalling of RNA polymerase leads to its dissociation from the DNA template, terminating the transcription process
  • Rho-independent termination does not require any additional proteins or factors, as the termination signal is encoded within the DNA sequence itself
• Comparison of the two mechanisms
  • Both Rho-dependent and Rho-independent termination result in the dissociation of RNA polymerase from the DNA template and the release of the newly synthesized RNA molecule
  • Rho-dependent termination requires the participation of the Rho protein and the hydrolysis of ATP, while Rho-independent termination relies solely on the formation of a specific RNA secondary structure
  • Rho-dependent termination can occur at various locations downstream of the gene being transcribed, whereas Rho-independent termination occurs at specific sites encoded by the DNA sequence

RNA Polymerase and DNA Strands in Transcription

• RNA polymerase is composed of two main components:
  • Core enzyme: Contains the catalytic subunits responsible for RNA synthesis
  • Holoenzyme: The complete RNA polymerase complex, including the core enzyme and sigma factor
• During transcription, RNA polymerase interacts with two important DNA strands:
  • Template strand: The DNA strand that is read by RNA polymerase to synthesize the complementary RNA molecule
  • Coding strand: The non-template DNA strand that has the same sequence as the RNA transcript (except for thymine being replaced by uracil)
• Nucleotides are the building blocks of RNA, added one by one to the growing RNA chain during elongation