Gene Expression: Prokaryotic Transcription

Gene Expression: Transcription in Prokaryotes

• RNA is a polymer of nucleotides joined by phosphodiester bonds
• RNA contains ribose sugar, uracil instead of thymine, and a hydroxyl group on the 2' carbon
• DNA nucleotides contain deoxyribose sugars, lack a free hydroxyl group on the 2' carbon, and are more stable
• RNA is less stable than DNA under alkaline conditions due to the free hydroxyl group
• RNA can have secondary structures due to hydrogen bonding between complementary bases
• RNA molecules perform various functions in cells such as ribosomal RNA (rRNA) that makes up the ribosome for protein assembly, messenger RNA (mRNA) carrying the coding instructions from DNA to ribosomes for protein synthesis, and transfer RNA (tRNA) that links mRNA codons to amino acids

Classes of RNA

• Ribosomal RNA (rRNA) forms the ribosome, the site of protein synthesis, and is found in both bacterial and eukaryotic cells
• Messenger RNA (mRNA) carries the coding sequence from DNA to the ribosome in both bacterial and eukaryotic cells
• Transfer RNA (tRNA) links codons in mRNA to specific amino acids in both bacterial and eukaryotic cells
• Small nuclear RNAs (snRNAs) are involved in processing pre-mRNA into mRNA in eukaryotic cells
• Small nucleolar RNAs (snoRNAs) are involved in processing rRNA in eukaryotic cells
• MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are involved in RNA interference (RNAi) in eukaryotic cells. RNA interference regulates gene expression by degrading mRNA or inhibiting translation

The Concept of Transcription

• All cellular RNAs are synthesized from DNA templates during transcription
• During replication, all nucleotides in DNA are copied
• During transcription, only small parts (single genes or a few genes) of the DNA molecule are transcribed into RNA.
• Transcription is a highly selective process; individual genes are transcribed only as needed or when their products are needed
• Transcription requires DNA template, raw materials (substrates) to build RNA molecules, and the transcription apparatus

The Template for Transcription

• The template for RNA synthesis is a single strand of the DNA double helix
• The transcription of a gene takes place on one of the two DNA nucleotide strands
• The nucleotide strand used for transcription is called the template strand.
• The non-template strand is not ordinarily transcribed and is also referred to as the coding strand, which has the same sequence as the RNA molecule, except that it replaces thymine (T) with uracil (U)

The Transcription Unit

• A transcription unit is a DNA segment that encodes an RNA molecule.
• Each transcription unit contains a promoter, RNA-coding region, and a terminator.
• The promoter is a DNA sequence that the transcription apparatus recognizes and binds.
• The RNA-coding region is a DNA sequence that is copied into an RNA molecule.
• The terminator is a DNA sequence that signals where transcription ends.
• The sequence of the non-template strand is written with the 5' end on the left and 3'end on the right, while the sequence of the RNA transcript proceeds in the 5'-3' direction.

The Transcription Apparatus

• The RNA polymerase carries out all the required steps of transcription.
• RNA polymerase is enhanced by various accessory proteins during transcription
• Bacterial cells typically possess one type of RNA polymerase responsible for all classes of bacterial RNA (mRNA, tRNA, and rRNA).
• RNA polymerase is a large multimeric enzyme containing polypeptide chains
• The core enzyme binds with a sigma factor to form a holoenzyme during transcription.
• The sigma factor controls RNA polymerase binding to the promoter, and initiates transcription at a proper start site.
• Eukaryotic RNA polymerase are large multimeric enzymes, consisting of more than a dozen subunits.
• Eukaryotic cells have three distinct types of RNA polymerases.
  • RNA polymerase I transcribes large rRNA (ribosomal RNA).
  • RNA polymerase II transcribes pre-mRNAs (pre-messenger RNA) along with some snoRNAs, miRNAs, and snRNAs.
  • RNA polymerase III transcribes tRNAs and small rRNAs along with some miRNAs, and snRNAs
• RNA polymerase IV transcribes some siRNAs in plants

The Process of Bacterial Transcription

• Transcription occurs in three stages:
  • Initiation: Transcription apparatus assembles and begins RNA synthesis on the promoter
  • Elongation: RNA polymerase unwinds the DNA, adding nucleotides to the 3' end of the growing RNA
  • Termination: Recognition of the end of the transcription unit, ending RNA separation from the DNA template

Initiation

• A consensus sequence is stretches of nucleotides within the DNA found in the promoter region.
• The common consensus sequence in bacterial promoters is centered about 10 bp upstream of the start site, and is called the -10 or Pribnow box.
• The -35 consensus sequence is located ~35 bp upstream of the start site.
• A promoter is a DNA sequence adjacent to a gene and required for transcription

Elongation

• RNA polymerase moves along the template DNA from 3' to 5', producing the RNA transcript by adding nucleotides to the 3' end of the growing RNA.
• Transcription takes place within a short stretch of DNA (~18 nucleotides) called the transcription bubble.

Termination

• RNA polymerase transcribes a specific terminator sequence
• Transcription stops after the terminator has been transcribed
• RNA polymerase stops synthesizing the RNA molecule , detachs from the DNA template and releases the RNA transcript.