Microbial Genomes and DNA structure

Microbial Genomes

• The genetic elements of microbes include chromosomal DNA, plasmids, and transposable elements.
• Prokaryotes follow the central dogma, utilizing informational macromolecules like eukaryotes.
• Typical prokaryotic genomes range from 4-8 Mb, coding for 4,000-8,000 genes (ORFs).

Bacterial & Archaeal DNA

• DNA is double-stranded
• Nucleotides are the nitrogenous bases, either purines (G and A) or pyrimidines (C and T).
• Nucleotides are linked by phosphodiester bonds.
• Strands are complementary and run antiparallel (5' to 3').
• Chargaff’s rule applies.
• The DNA structure folds into a double helix.

Genome Organization

• Prokaryotes typically have one circular dsDNA chromosome, with some exceptions.
• Chromosomes are condensed into negatively supercoiled domains, facilitated by DNA gyrase.

Microbial Genetic Elements

• Chromosomes are large, ranging from 4-8 Mb.
• Chromosomes contain essential ("housekeeping") genes.
• Chromosomes have one copy per cell.
• Chromosome replication is strongly regulated by the cell cycle.
• Plasmids are small, ranging from 1-1000 kb.
• Plasmids contain "bonus" genes.
• Plasmids can have one to hundreds of copies
• Plasmid replication is independent of the chromosome.

DNA Replication

• Bacterial DNA replication follows a semi-conservative model.
• Replication starts at an origin, proceeding in both directions.
• This is referred to as theta (θ) replication.
• DNA replication enzymes form a replisome.

Transcription - Initiation

• Sigma factors are prokaryotic transcription factors, with σ70 used for "housekeeping" genes.
• DNA consensus sequences bind at promoters which are upstream of transcription sites.
• The -10 region (Pribnow box) and -35 region are part of this.
• Variations in consensus sequences influence transcription rates.

Transcription - Elongation

• RNA polymerase binds σ70, at the promoter, creating the RNA polymerase holoenzyme.
• σ70, dissociates after initiation, then the RNA polymerase core enzyme transcribes DNA from 3' to 5', adding RNA nucleotides from 5' to 3'.

Transcription - Termination

• Inverted repeats in DNA leads to the formation of a stem-loop.
• Termination is followed by a stretch of adenines on the template strand.
• RNA polymerase stalls and detaches from the DNA.

Operons and Polycistronic mRNA

• Genes for related enzymes are in an operon.
• Multiple genes are sequentially ordered, controlled by a single promoter.
• Genes are co-transcribed into a single transcript, or polycistronic mRNA.

Transcriptional Regulation

• Transcription factors are allosteric proteins that regulate transcription rate.
• Activator proteins turn transcription "on" and bind upstream of the promoter at the activator binding site.
• Repressor proteins turn transcription "off" and bind downstream of the promoter at the operator.
• Effector molecules control the action of transcription factors when binding to DNA.
• Inducers bind to activator proteins.
• Corepressors bind to repressor proteins.

Transcriptional Regulation: Enzyme Repression

• Genes are expressed only if the end-product of a biochemical pathway is absent.
• Genes are normally "on" without anything bound to the operator.
• Arginine acts as a corepressor, binding to a repressor protein.
• The corepressor-bound repressor binds DNA at the operator, for arginine synthesis.
• RNA polymerase cannot transcribe the gene with a repressor at the operator.
• Arginine detaches from the repressor if the cell runs out of arginine, transcription starts, and the enzymes are produced.

Transcriptional Regulation: Enzyme Induction (Derepression)

• Genes are expressed only if a substrate molecule is present.
• Genes are normally "off" with a repressor bound to the operator.
• Lactose acts as an inducer, binding to the repressor protein.
• The inducer-bound repressor detaches from the lactose-metabolizing region.
• RNA polymerase transcribes with the repressor off the operator, with the cell metabolizing the lactose.
• Lactose detaches from the repressor if the cell runs out of lactose, the repressor binds to the operator, and transcription stops.

Transcriptional Regulation: Enzyme Activation

• Some genes are expressed rarely, they have weak promoters.
• Maltose acts as an inducer, binding to an activator protein.
• The inducer-bound activator binds DNA at an activator upstream from the promoter.
• Sigma factor/RNA polymerase binding is improved with DNA-bound activators, with transcription occuring, and the cell metabolizing maltose.
• Maltose detaches from the inducer if the cell runs out of maltose, which causes it to detach from the activator site, and transcription stops.

Translation 1

• Prokaryotic translation is virtually identical to eukaryotic translation.
• Ribosomes attach to mRNA at RBS (Shine-Dalgarno), starting translation at the start codon (AUG).
• tRNAs bring amino acids, based on complementary codon-anticodon.
• Translation ends at a stop codon (UAA, UAG, UGA).
• Key differences in mRNA:
  • Polycistronic mRNA has multiple RBS.
  • Prokaryotic mRNA requires no processing. -It has no 5' cap, poly-A tail, or introns.

Translation 2

• The differences include:
  • 70S ribosomes are used.
  • First amino acid is formylmethionine (fMet).
  • Codon bias varies among species.
  • Not all ORFs in polycistronic mRNA share the same reading frame.
  • Some species use stop codons for selenocysteine (Sec) and pyrrolysine (Pyl).

Coupled Transcription/Translation

• Prokaryotes do not have a nucleus.
• mRNAs do not require they export before translation.
• Translation begins before transcription ends.
• Prokaryotic polysomes have multiple ribosomes on each transcript.

Protein Processing

• Post-translational changes are sometimes required:
  • Chaperones aid protein folding and cofactor adding.
• Proteins may need specific place targeting.

Translational Regulation: sRNA

• sRNA is a single-stranded RNA that is 40-400 nt in length.
• Alters rates of translation.

Translational Regulation: Riboswitches

• The aptamer within the 5'UTR region of mRNA generates secondary structures based on the absence or presence of a signal metabolite.

Transcriptional Regulation: Attenuation

• Operates solely in prokaryotes.
• Relies on transcription and translation coupling.
• The mRNA's 5' end creates either of two stem-loops through transcription.
• Translation speed of the mRNA leader determines the final stem-loop.