Microbial Regulation & Genetics

• Regulation is a sophisticated process involving genetic and metabolic factors to adapt and maintain resilience in microorganisms.
• Regulation occurs through constitutive/responsive genes at multiple levels including DNA structure and post-transcription.
• Key regulatory parts are cis/trans elements.
• Regulatory proteins include positive and negative controls, global regulators, catabolite repression, and two-component sensor kinases.
• Attenuation and varying forms of Regulatory RNAs are also key.

Glossary

• Jacques Monod researched the subject of "enzymatic adaptation," leading to the concept of "induced enzyme synthesis".
• Mixotrophic growth refers to growth using two carbon sources.
• "-omics" includes proteomics, transcriptomics (proteins vs. RNAs, tandem mass spec (MS) vs. RNA seq (illumina).
• Constitutive genes/proteins/RNAs are required under all growth conditions, acting as housekeeping genes for central metabolic pathways.
• Responsive genes are expressed only under certain conditions.
• Global regulators oversee genes/operons across multiple pathways.
• Cis-elements and trans-factors regulate gene expression.
• Effectors (inducers, co-repressors, inhibitors), Lac-operon structure and regulatory elements within LacI are key concepts.
• Catabolite repression involves CRP and cAMP regulation.
• Attenuation controls transcription.
• Regulatory RNA, including sRNA, RNAT, and riboswitches all control gene function.
• Hfq protein function related to sRNA activity.
• Ligands participate in regulatory molecular interactions.

How Are Regulatory Mechanisms Understood?

• Jacques Monod researched the diauxic growth of E. coli in 1942 on a mixture of glucose and lactose,
• This showed "enzymatic adaptation."
• Only a subset of genes is expressed at any given environmental condition.
• Transcription produces mRNA from DNA and translation produces proteins from mRNA.
• Omics tools help us study mechanisms.
• RNA-seq (Illumina) is used to measure transcriptomics
• Tandem mass spectrometry (MS/MS) is used to measure proteomics.

Constitutive vs Responsive Genes

• Constitutive genes code for proteins/RNAs always needed for growth, including housekeeping genes and central metabolic pathways.
• Responsive genes are expressed only under certain conditions, such as stress, temperature, other metabolic conditions, biofilm formation, growth vs dormancy, and nutrient availability

Why Regulation

• Regulation conserves energy and resources, but minimizing unnecessary transcription and translation.
• Regulation balances contradictory cellular functions.
• RNA and protein synthesis uses approximately half of a cell's energy.
• Biosynthesis includes nucleotides (NTPs) and amino acids (AA).
• Polymerization is costly.

Decision Points and Regulation

• Decision points are throughout microbial life.
• Contradictory cellular functions must be controlled, such as;
• The steps of Sporulation like vegetative growth, endospore formation, and germination
• Forespore development includes layers of peptidoglycan and coat proteins.

Levels of Molecular Regulation

• Regulation can be at the level of DNA structure/arrangement, including phase variation and methylation.
• Regulation can be transcriptional, involving DNA-binding proteins and attenuation.
• Translational regulation includes riboswitches, small RNA, and RNA stability/degradation.
• Post-translational modifications also play a role.

Regulatory Elements

• Cis-regulatory elements act from the same molecule as DNA sequences near a gene include promoters, RBS, terminators and operators.
• Trans-acting factors act from a different molecule from factors that bind the cis elements.
• Trans-acting factors include RNA polymerase, sigma factors, repressors, and activators.

Genetic Organization

• Gene clusters are physically adjacent on DNA and are co-transcribed.
• Operons feature two or more genes transcribed into a single RNA.
• Regulons comprise a set of genes/operons under the same regulatory element.
• Global regulators affect genes/operons across diverse cellular pathways.
• Specific regulators control a small set of genes.

Regulatory Proteins

• Transcription initiation is a major site of regulatory control in bacterial cells.
• Aspects to understand this control are DNA-binding regulatory proteins act as at the gene promoter and environmental changes impact regulatory protein activities.
• Repressors bind DNA and prevent transcription.
• Activators bind DNA and activate RNA-pol binding.

Transcriptional Control

• In negative control of transcription, RNA polymerase can become blocked with the help of repressors.
• The operator is a specific region on DNA to which the repressor protein binds, preventing transcription.
• Effectors include inducer-small molecules that can induce an activator.
• Alternatively effectors can include co-repressors that repress a repressor.
• In positive control of transcription, an activator facilitates RNA polymerase recognition of the promoter.

Genetic Circuits

• Genetic circuits can be composed of factors such as:
• Oxygen's effects on gene for aerobic (FNR) growth.
• The relationship between glucose and genes for lactose utilization.
• The relationship between Tryptophan and tryptophan biosynthesis genes.

Environmental Sensing

• Changes in extracellular conditions are detected by plasma membrane-embedded signaling molecules to influence gene expression.
• Two-component systems rely on protein phosphorylation
• A sensor kinase is membrane-bound and a response regulator is a cytosolic protein.

Summary of Key Concepts

• Bacterial genomes handle environmental contingencies and cells do not express all genes maximally under all conditions.
• Microbes sense their internal and external environments for specific protein synthesis.
• Gene regulation occurs at multiple levels.
• Regulatory proteins help the cell sense and react, with two-component systems sensing the external environment.

Studying Regulatory Proteins

• Study regulatory proteins through:
• Mutations (knockouts)
• Gel-shift assays (protein DNA binding assays).
• DNase footprinting assays (protein binding site assays)
• Transcriptional start mapping.
• Fusions relating to transciption and translation.

Studying Regulatory Proteins

• Electrophoretic mobility shift assay (EMSA) can be used to assay by DNA binding.
• DNase footprinting can show the binding site of the protein.
• Transcriptional start mapping provides information for transcription.
• Transcriptional reporter-fusion assay allows scientists to monitor gene expression.

The Lac Operon and Catabolite Repression in Detail

• In Lac operon regulation, LacI protein mRNA products are regulated, the operator is where LacI binds, and the lacZ lacY and lacA products are utilized for lactose digestion.
• LacI acts as repressor, becoming inactive and active under different regulatory regimes.
• In the absence of lactose, LacI repressor binds to the operator, blocking transcription.
• With lactose present, allolactose binds to LacI, inactivating it, enabling transcription
• Lactose is not utilized when glucose is present.
• Catabolite repression is the suppression of alternative metabolic pathways that are replaced by a preferred carbon and/or energy source.
• In catabolite repression CRP activates the CRP protein; global regulators that can signal transcription of many catabolic genes by binding cAMP
• When the Lac-operon is on allolactose binds to LacI repressor and cAMP-CRP activator are bound to the promoter, assuming glucose is absent

More Lac Operon Details

• PTS facilitates phosphorylated glucose transport in cells.
• With glucose, the PTS transfers and phosphorylates glucose, inhibiting lactose uptake, where LacI keeps the lac-operon OFF.
• Without glucose, cAMP is produced and activate the adenylyl cyclase, stimulating lac enzyme synthesis .
• Lactose becomes present and Lactose is active.

Attenuation of the Trp Operon

• In attenuation, regulation can be determined by translation.
• Attenuation is a 2 stage process; first a simple On/Off switch, the second is a modulation system with a leader
• In bacteria, transcription and translation are coupled.
• Translation relies on available amino acid pools and transcription can be terminated.
• An intrinsic terminator loop or anti-attenuator loop (if tryptophan level is low) can be created on the trp codons depending on its utilization.
• Where tryptophan levels are high the ribosome will stop covering mRNA regions and create an intrinsic terminator and attenuate.
• When tryptophan levels are low the ribosome will stall and create the anti-attenuator loop and allow the gene to continue to be transcribed.

Regulatory RNAs: small RNA

• Regulatory RNAs (trans-) prevent translation/control stability
• Regulatory RNAs (cis-) prevent transcription/control stability
• Small RNA are around 50-300 nucleotides.
• Hfq proteins act as chaperones
• Small RNA requires specific base pairing sequences.
• Regulatory RNAs binding to the mRNA affects their regulation via the RBS site.
• The small RNA can direct RNase E

Regulatory RNAs: Riboswitches, Zippers, and RNATS

• Regulatory elements are in an mRNA.
• Structure is dependent on its 3D structure.
• Structure changes, in turn, alter the transcription and translation of specific mRNA.
• The small RNA requires a specific base pairing sequence
• Small RNA binds to the RBS site and/or causes translational changes in the structure.
• RNA thermometers consist of two mutually exclusive structures, dependent on temperatures.

The Broad Effects of Translational Regulation

• Translation enhancers use stem loops to stablize ribosomes.
• Shine-Dalgarno sequences are known to sequester ribosomes
• Proteins can also sequester the ribosomes.

Summary

• The lac-operon: operon is off when lacI binds, on when there is allolactose.
• Tryptophan operon is regulated by repression.
• Regulatory RNAs stabilize or destabilize mRNA.
• Includes riboswitches and RNATS.