RNA Polymerase Functions

RNA Polymerase I Functions

• Searches for DNA initiation sites (promoters)
• Unwinds a short stretch of double helical DNA to give single-strand DNA templates
• Selects correct ribonucleoside triphosphates and catalyzes phosphodiester bond formation
• Detects termination signals
• Interacts with activator and repressor proteins to modulate transcription

Differences between RNA Polymerase and DNA Polymerase

• RNA polymerase does not require a primer, as RNA chains can be formed de novo
• RNA polymerase does not have nuclease activity, resulting in low fidelity that can be tolerated
• DNA template is fully conserved in RNA synthesis, but semi-conserved in DNA synthesis

Structure of Prokaryotic RNA Polymerase

• RNA polymerase of E. coli has a specific structure

Bases in DNA and RNA

• Adenine (A) is present in both DNA and RNA
• Cytosine (C) is present in both DNA and RNA
• Guanine (G) is present in both DNA and RNA
• Thymine (T) is present in DNA, replaced by Uracil (U) in RNA

Classes of Prokaryotic RNA

• Ribosomal RNA (rRNA): 16S, 23S, and 5S
• Transfer RNA (tRNA)
• Messenger RNA (mRNA)

Structure of Prokaryotic Messenger RNA

• Shine-Dalgarno sequence facilitates initiation of protein synthesis
• Sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA

RNA Transcription

• Synthesis of RNA from DNA
• RNA synthesis grows from 5' end to 3' end
• Initiation of protein synthesis is facilitated by the Shine-Dalgarno sequence

Types of RNA

• Eukaryotic RNA: mRNA, tRNA, rRNA, and hnRNA
• mRNA bears the genetic code (5%)
• tRNA transfers amino acids to ribosomes (15%)
• rRNA is responsible for protein synthesis (80%)
• hnRNA is nuclear RNA before processing

Transcription Process

• Closed promoter complex forms
• Open promoter complex forms
• Initiation of transcription occurs
• Elongation of RNA transcript occurs
• Termination of transcription occurs
• RNA product is formed

Elongation of RNA Transcript

• Nascent RNA forms a hybrid helix with the template DNA strand
• After several nucleotides are added to the growing RNA chain, the nucleotide at the 5' end of the RNA chain becomes unpaired
• RNA polymerase loses the σ subunit
• NusA protein aids the elongation and binds to the RNA polymerase

coli RNA Polymerase

• The coli RNA polymerase is a multisubunit protein composed of subunits a, b, b', and s.
• Subunit a: 2 units, role uncertain
• Subunit b: 1 unit, forms phosphodiester bonds
• Subunit b': 1 unit, binds DNA template
• Subunit s: 1 unit, recognizes promoter and facilitates initiation

Eukaryotic RNA Polymerases

• There are three types of RNA polymerases in eukaryotes: I, II, and III
• RNA polymerase I: localized in nucleolus, transcribes 18S, 5.8S, and 28S rRNA
• RNA polymerase II: localized in nucleoplasm, transcribes mRNA precursors and snRNA
• RNA polymerase III: localized in nucleoplasm, transcribes tRNA and 5S rRNA

Template and Coding Strands

• The strand that is transcribed into an RNA molecule is referred to as the template strand (or antisense strand)
• The other DNA strand is referred to as the coding strand (or sense strand)
• A given strand of double helical DNA can serve as a template strand for some genes and a coding strand for other genes

Transcription of Genes from Both Strands

• Genes can be transcribed from both strands of DNA
• The template strand is always read in the 3' to 5' direction
• The coding strand is identical except that U replaces T in the mRNA transcript

Transcription Initiation

• The sigma subunit binds to the -10 region, forming a closed promoter complex
• The holoenzyme has a high affinity for promoter regions because of the sigma factor
• Once initiation takes place, RNA polymerase does not need a high affinity for the promoter, and the sigma factor dissociates from the core polymerase

Transcription Bubble

• The transcription bubble is the region containing RNA polymerase, DNA, and nascent RNA
• It is a locally melted (or "bubbled") region of DNA

Termination of Transcription

• Formation of phosphodiester bonds ceases
• The RNA-DNA hybrid dissociates
• The melted regions of DNA rewind to form a double helix
• The core enzyme (devoid of s) has a low affinity for double helical DNA, so RNA polymerase leaves the DNA
• The sigma factor rejoins the core enzyme, reforming the holoenzyme, which can repeat the transcription process

RNA Processing

• tRNA and rRNA molecules are much smaller than the primary transcripts
• Modification of bases and ribose in RNA occurs, such as methylation of bases in prokaryotes and methylation of ribose units at the 2'-OH end in eukaryotes

Processing of tRNA in Eukaryotes

• The tRNA precursor undergoes a series of alterations to form mature tRNA:
  • Cleavage of the 5'-leader sequence
  • Splicing to remove an intron
  • Replacement of the 3'-terminal UU by CCA

Transcription in Eukaryotes

• Transcription of mRNA in eukaryotes is similar to that in bacteria, but with notable differences:
  • Eukaryotic cells contain 3 classes of nuclear RNA polymerase
  • Transcription in eukaryotes occurs in the nucleus, while translation occurs in the cytosol
  • In mRNA, both the 5' and 3' termini are modified
  • Splicing: introns are spliced out of mRNA precursors
  • Enhancers: nucleotide sequences that increase the utilization of eukaryotic promoters