Microbial Genomes: DNA, Plasmids, and Organization

• Includes chromosomal DNA, plasmids, and transposable elements
• Prokaryotes follow the central dogma, using the same informational macromolecules as eukaryotes

Microbial Genomes

• The entire set of genetic elements in a microbe is the microbial genome

Genome Size

• Typical genome size ranges from 4-8 Mb
• Genomes code for 4000-8000 genes (ORFs)

Bacterial and Archaeal DNA

• Double-stranded DNA is present
• Nucleotides are purines (G and A) and pyrimidines (C and T)
• Nucleotides are linked via phosphodiester bonds
• Strands run anti-parallel and are complementary (5' -> 3')
• Follows Chargaff's rule
• Folds into a double helix

Genome Organization

• Prokaryotes usually have one circular dsDNA chromosome
• Chromosomes compact into negatively supercoiled domains using DNA gyrase

Chromosome vs Plasmid

• Chromosomes are large, ranging from 4-8 Mb while Plasmids are small 1 - 1000 kb
• Chromosomes are essential for "housekeeping" genes while Plasmids contain "bonus" genes
• Chromosomes exist in one copy per cell while Plasmids range from one to a hundred copies
• Chromosomic replication is strongly regulated by the cell cycle and Plasmid replication is independent from chromosomes

DNA Replication

• Uses a semi-conservative model
• Replication begins at an origin of replication and proceeds in both directions, referred to as theta (θ) replication
• DNA replication enzymes group to form the replisome

Transcription Initiation

• Prokaryotic transcription factors are sigma factors, with σ70 for "housekeeping" genes
• DNA consensus sequences bind at promoters located upstream of transcription initiation sites
• The -10 region (Pribnow box) and -35 region are key
• Variations in consensus sequences control transcription frequency

Transcription Elongation

• RNA polymerase binds σ70 at the promoter, forming the RNA polymerase holoenzyme
• The RNA polymerase core enzyme transcribes DNA in a 3' to 5' direction (adding RNA nt 5' to 3') after σ70 dissociates

Transcription Termination

• Inverted repeats (DNA) form a termination stem-loop, followed by stretch of adenines on the template strand
• RNA polymerase stalls and "falls off" the DNA

Operons and Polycistronic mRNA

• Genes for related enzymes are sometimes organized into an operon
• Multiple genes are sequentially ordered and controlled by a single promoter
• Genes are co-transcribed to produce a single transcript, known as polycistronic mRNA

Transcriptional Regulation

• Transcription factors are allosteric proteins controlling transcription rate

Activator Proteins

• Activator proteins turn transcription "on"
• Activator proteins bind to an activator binding site upstream of the promoter

Repressor Proteins

• Repressor proteins turn transcription "off"
• Repressor proteins bind to an operator downstream of the promoter

Effector Molecules

• Effector molecules allow or prevent transcription factors from binding DNA
• Inducers bind to activator proteins
• Corepressors bind to repressor proteins

Enzyme Repression

• Genes are expressed when the end-product of a biochemical pathway is absent
• Genes are normally "on" without anything bound to the operator
• Arginine acts as a corepressor and binds to a repressor protein
• Arginine-bound repressor protein binds DNA at the operator region of arginine-synthesis genes
• RNA polymerase cannot transcribe the gene when a repressor is attached to the operator, therefore the cell stops producing arginine
• Arginine will fall off the repressor, operator, allowing transcription to begin if the cell runs out of arginine

Enzyme Induction (Derepression)

• Genes are expressed when a substrate molecule is present
• Genes are normally "off" with a repressor bound to the operator
• Lactose acts as an inducer and binds to the repressor protein
• The inducer-bound repressor protein falls off the operator
• Lactose metabolizes when the RNA polymerase begins transcription without the repressor
• Transcription stops, lactose falls of the repressor, and binds to the operator when a cell runs out of lactose

Enzyme Activation

• Some genes are expressed rarely or in very small amounts because they have weak promoters
• Maltose acts as an inducer and binds to an activator protein
• The inducer-bound activator protein binds DNA at an activator binding site upstream of the promoter
• DNA-bound activators improve sigma factor/RNA polymerase binding, transcription occurs, and Maltose is metabolized
• Maltose falls off the inducer, therefore falling off the activator binding site when a cell runs out of Maltose, therefore transcription stops

Translation

• Translation is virtually identical to eukaryotic translation
• Ribosomes bind mRNA at the Shine-Dalgarno sequence (RBS) and starts translation at a start codon (AUG)
• Elongation proceeds as tRNAs bring amino acids by complementary codon-anticodon interactions
• Translation ends at a stop codon (UAA, UAG, UGA)

Eukaryotic Translation Differencess

• mRNA is polycistronic with multiple RBS
• Prokaryotic mRNA does not require processing unlike Eukaryotic translation
• Does not need a 5' cap
• Does not need a poly-A tail
• Does not need processing of introns

Prokaryotic Translation Differences

• Uses 70S ribosomes
• The first amino acid is formylmethionine (fMet)
• Codon bias varies among species
• ORFs in polycistronic mRNA may have different reading frames
• Selenocysteine (Sec) and pyrrolysine (Pyl) can be encoded by "stop codons" in some species

Coupled Transcription/Translation

• Prokaryotes do not have a nucleus
• mRNAs are not exported before translation begins
• Translation begins before transcription has ended
• Prokaryotic polysomes also have multiple ribosomes on each transcript

Protein Processing

• Requires assistance during post-translational modification to add multi-subunit structures as well as other cofactors
• Proteins are targeted to specific locations

Translational Regulation

• Small RNA's are 40-400 nt long that pair with segments of mRNA to alter the rate of translation

Riboswitches

• Riboswitches are a part of translation that include the Aptamer region within the 5'UTR of mRNA that alternate secondary structure depending on the absence or presence of a signal metabolite

Transcriptional Regulation: Attenuation

• Unique to prokaryotes
• Is possible due to the coupling of transcription and translation
• During transcription the 5' end of mRNA determines which type of stem loop to form of which there are two possibilities
• The speed of the translator determines which stem loop will form

At First There Was Tryptophan

• If the cell has Trp, the 5' mRNA end is translated quickly (regions 1/2 are blocked)
• A haripin is formed from regions 3 and 4 which terminates transcription

And Then There Was None

• Translation halts, blocking region one is the cell lacks Trp
• Regions 2 and 3 form a hairpin that permits transcription and translation to proceed