MICB 211 Chapter 9 Notes

Signal Transduction

-prokaryotes can sense changes in ______ of their environment

  • chemical gradients

  • pH

  • osmolarity

  • light

  • magnetic fields

-prokaryotes have the ability to respond to these sensations

-different sending-response systems act over different time scales

-some senstationst rigger immediate change in behaviour without synthesis of any new proteins

  • others require a change in gene expression because new proteins are required to respond to the sensation

One and Two Component Systems

-sensing and responding to environment involve protein-based signal transduction systems

-2 kinds of signal transduction systems

-more one-component systems than two-component systems in bacteria

-for both component systems:

  • responses are specific dependent on the function of the output domain

-3 broad classes of output domains

  1. enzymatic activity

  2. bind to other proteins

  3. bind to DNA to affect transcription/gene expression

Domain

  • signal transduction protein composed of parts

One-Component System

-single protein serves 2 functions

  1. domain detects signal (senor kinase)

  2. domain initiates the cellular response (response regulator)

-signal received directly by binding of small chemical from environment to sensor kinase

  • sensor kinase usually embedded in plasma membrane

-signal activates its kinase activity → phosphorylate the response regulator to change its conformation so that it can actively bind to DNA (promoter region)

-triggers a change in gene expression to either promote production of proteins needed to respond to sensation or even turn off expression of proteins that be making it respond negatively to changes in environment

-response regulator is physically attached to the sensor (membrane bound)

  • entire system is constrained to the membrane

  • system has to navigate around the membrane and/or DNA target has to shift to meet the response regulator

Two-Component System

-2 domains are separate proteins

-sensor detects the signal and interacts with second protein (response regulator)

-response regulator is free-floating in cytoplasm

  • able to actively find and bind to its target

  • elicits a faster response because it’s not constrained

Knock-Out Mutants and Complements

Laboratory Plasmids

  • origin of replication (Ori): allows plasmid to replicate independently of chromosome

  • often 2 selection/selectable markers: allows researchers to selectively grow the cells that have taken up the plasmid

  • multiple cloning sites (MCS): regions where researchers can insert a target gene and/or promoter region

  • expression plasmids

    • produces a protein of interest; protein of interest doesn’t need to be bacterial in nature

  • recombinant plasmids

    • plasmid that has another species protein cloned in it

    • normally have MCS in front of existing promoter

  • reporter plasmids

    • to not express a gene of interest but determine whether a gene of interest is being expressed

    • promoter of gene of interest is cloned in front of reporter protein where a read-out can be easily measured

    • ex: GFP derived from jellyfish or lacZ (beta-galactosidase) enzyme

Knock-Out Mutants

-laboratory derived strains where researchers have deleted a gene of interest to see what happens to the phenotype being studied

ex: researchers interested in seeing if fliC (involved with flagellar biosynthesis, swimming motility) → researchers knock-out (remove) fliC → look for absence or inhibition of swimming

-if gene is essential for that phenotype → expect phenotype to be attenuated or absent in mutant compared to wild type

Complements

-deleting a gene from the chromosome can have unforeseen effects on various elements

-polar effects

  • side effects that don’t contribute to phenotype loss seen in a knock-out mutant

-researchers complement mutant by reintroducing the original gene on an expression plasmid into the cell

-goal is to see if phenotype can be restored by expressing the original gene in isolation

  • if phenotype restored → confidently attribute that phenotype loss was due to that single gene deletion

  • if phenotype not restored → polar effects contributing to phenotype loss that may not necessarily be because of the target gene removed

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