Prokaryotic Transcription and Translation - Dr. Michael Green

Prokaryotic Genome Organisation

• Prokaryotes lack a nucleus surrounded by a complex nuclear membrane.
• They have a single large circular “chromosome” located in the ‘nucleoid’.
• They have smaller circular extrachromosomal DNA called plasmids.
• Prokaryotic genome is supercoiled.
• Most prokaryotes are haploid.
• They contain small amounts of non-coding DNA, with coding DNA organized into operons.

Central Dogma of Molecular Biology

• Protein synthesis occurs in two major steps: transcription and translation.
• Protein synthesis requires three major types of RNAs: mRNA, tRNA, and rRNA.
• The process is highly regulated, with not all genes expressed at the same time.
• The ribosome is the main translational unit that synthesizes the polypeptide.

Transcription

• The goal of transcription is to synthesize a chain of RNAs (mRNA) via RNA synthesis.
• Transcription occurs in three stages: initiation, elongation, and termination.
• RNA polymerase is the main transcriptional unit.
• Transcription begins when RNA polymerase binds to the promoter.
• The association of sigma factor dramatically increases binding affinity and specificity.
• Prokaryotic promoters have two characteristic features: TTGACA sequence and TATAAT sequence or Pribnow box.

Initiation of Transcription

• Holoenzyme first binds loosely to the -35 box in a closed complex.
• It then binds tightly to the -10 and the DNA unwinds, forming the open complex.
• RNA polymerase is then oriented correctly, sigma factor is released, and transcription proceeds at the next nucleotide.

Translation

• Translation occurs in three phases: initiation, elongation, and termination.
• Translation requires mRNA, ribosomes, tRNA, and amino acids as well as other helper proteins.
• The ribosome is a protein synthesizing machine composed of rRNA and ribosomal proteins.
• The ribosome has 3 active sites: A SITE, P SITE, and E SITE.
• EF-Tu and EF-G are other proteins that help the process along.

Initiation of Translation

• tRNA transfers the amino acid to the growing peptide chain.
• Its key features are a 3 amino acid attachment site and an anticodon.
• The correct amino acid must find the correct tRNA.
• Initiation factor proteins deliver the small subunit to the mRNA.

Elongation

• The ribosome begins to read along the mRNA strand, building a protein one amino acid at a time.
• EF-Tu and EF-G help this process along.

Termination

• Translation requires the recognition of the stop codon by release factors (RF1).
• This forces the ribosome to release the finished polypeptide sequence from the P site.
• Termination occurs in three steps: recognition of the stop codon, hydrolysis of the ester bond, and dissociation of RF1/RF2 with the help of RF3.

Regulation of Gene Expression

• Bacteria can regulate the expression of genes in response to external stimuli.
• This is achieved through two-component regulator systems (TCRS) which transduce signals from outside the cell to the nucleoid.
• TCRS’s consist of a membrane-bound sensor kinase and a DNA-binding response regulator.

The lac Operon

• The lac operon is a set of lactose metabolizing genes that are induced only in the presence of lactose.
• The operon consists of three genes: lacA, lacZ, and lacY.
• LacY is required for the movement of lactose into the cell via transmembrane protein transport.
• LacZ turns lactose into glucose and galactose which can now be used by the cell.