Microbiology Midterm 3

where to start? transcription initiation

• promotor- DNA upstream of a gene recognized and bound by RNA polymerase so it can transcribe the following gene

• transcription start site- area between the promotor and the gene where RNA polymerase begins transcribing, an untranslated region of DNA

• transcription stop site- area after the gene where RNA polymerase stops transcribing, an untranslated region of DNA

what is a promotor?

• consensus (conserved) sequences at -10 and -35 (nucleotide distance from transcription start site)

  • slight differences in their NT sequence can modulate the strength of promotors

• RNA Pol sigma factor

  • binds to the space in between the -10 and -35 sequences of the promotor and falls off of the RNA Pol once transcription begins

  • bigger number = bigger size

  • most bacteria have more than one type of sigma factor (heat-shock, motility, chemotaxis, etc.) meaning that they can use RNA to regulate gene expression

  • the most common sigma factor is RpoD sigma 70

transcription termination

• Rho-dependent termination: Rho (a hexamer DNA helicase) binds to C-rich regions in mRNA → RNA is threaded through Rho as it hydrolyzes ATP pulling it closer to the RNA Pol → contact between Rho and RNA Pol causes transcription to terminate

• Rho-independent termination: an mRNA hairpin blocks RNA Pol so it cannot move forward → the poly-U site of the hairpin weakly pairs with DNA so the mRNA can dissociate → NusA protein supports the hairpin and aids in mRNA dissociation

the ribosome

• composed of two subunits that each include rRNA and proteins (mostly rRNA)

• large subunit 50S + small subunit 30S = 70S ribosome

  • 30S small subunit contains a16S rRNA, 50S large subunit contains a 5S and a 23S rRNA

• makes peptide bonds between amino acids using the ribozyme peptidyltransferase (a catalytic RNA molecule, 23S rRNA in large subunit)

translation

• translation initiation

  • requires a Shine-Dalgarno (SD) site upstream of a start codon in the mRNA

    • a ribosomal binding site (RBS)

    • AGGAGG consensus sequence

    • 4-8 bases upstream of the start codon

    • helps recruit a ribosome for translation by base-pairing with a complementary sequence located at the 3’ end of the 16S rRNA in the small subunit

  • starts at AUG 95% of the time

• translation termination

  • stops when a ribosome encounters a stop codon

• cotranscriptional translation

  • ribosome binds to mRNA that is still being made by RNA Pol and begins translation while transcription is still occurring

  • possible since bacteria do not have a nucleus

transcription and translation in archaea

• transcription: more similar to that of eukaryotes

• translation: no SD sequence and starts with an unmodified Met (AUG)

protein folding

• many proteins spontaneously fold into their proper shape via interactions within themselves and with their environment, utilizing no energy

• other proteins require assistance from chaperones and energy

  • GroEL-GroES: barrel-shaped chaperone (GroEL = barrel, GroES = lid) that creates the proper environment for damaged or misfolded proteins to try folding again, can have 1 (bullet) or 2 (football) lids

  • DnaK and DnaJ: form clamps around unfolded or misfolded proteins to prevent them from aggregating and to either facilitate their proper folding or stabilize and deliver them to GroEL

  • if a protein cannot fold even after assistance, it is destroyed by a protease

protein secretion

• proteins meant for the cell membrane are tagged with hydrophobic N-terminal signal sequences of 15-30 amino acids

  • co-translational export (the Sec system): SRP binds to SS to pause translation of the peptide → FtsY binds to and delivers SRP-peptide-ribosome complex to SecYEG translocon → GTP hydrolysis by SRP transfers peptide-ribosome complex to SecYEG and translation resumes —> SS released by translocon into cell membrane via a lateral channel

• secretion into the periplasm

  • SecA-dependent secretion pathway: SecB recognizes SS, keeps completely synthesized peptide unfolded and delivers it to SecA-SecYEG complex → SecA binds to SS and pushes peptide into SecYEG translocon using ATP → translocon releases SS into membrane and SS is cleaved by its signal peptidase → PMF drives complete translocation of protein

  • twin arginine translocase (TAT) system: protein is translated and folded → TatB-TatC complex recognizes short twin-arginine-containing SS (RRXFXK) and the protein binds to it so it can then begin recruiting TatA monomers → TatA monomers assemble into translocon using PMF → translocon transports protein into periplasm and its SS is cleaved by its signal peptidase → TatA channel dissociates and the system resets

bacterial gene regulation

• bacterial cells can differentiate to adapt to their environment by regulating their gene expression

  • example: cyanobacteria differentiate into 2 different types- heterocysts express nitrogen fixation genes, other expresses Z pathway genes for carbon fixation

• bacteria are logic machines: input = glucose/lactose → compute → output = express genes utilizing glucose/lactose

• a bacterial genome contains 1000s of different proteins

• bacterial are frugal → they do not make proteins unless necessary and can control gene expression at various levels

  • regulation at transcription level is energy-efficient but slow, regulation at translation level is fast but costly (in terms of energy and resources)

regulation of transcription

• most regulation is done during transcription initiation

  • promotors vary in strength depending on their sequence: weak = less RNA Pol binding and less gene copies, strong = more RNA Pol binding and more gene copies

  • repressor: DNA-binding protein that binds to operator sequence downstream of the promotor, blocking RNA Pol so gene expression is reduced/downregulated

  • activator: DNA-binding protein that binds upstream of the promotor, recruiting RNA Pol to the promotor and increasing the strength of its binding so gene expression is increased/upregulated

  • activators and repressors: bind to specific sites, are either dimers or tetramers, recognize palindromic sites (inverted repeats, 2 binding sites for a dimer), and have their DNA-binding ability modulated when ligands bind to them

    • inducers: ligands that bind to repressors so they release from their binding and the gene is expressed and bind to activators so they bind to their DNA site and the gene is expressed

    • corepressors: ligands that bind to repressors so they bind to their operator and the gene is not expressed and bind to activators so they release from their binding and the gene is not expressed

E. coli lactose operon

• operon: a unit of many functionally related genes that is co-transcribed and co-regulated

• import lactose into cell via symporter lactose permease (LacY) → beta-galactosidase (LacZ) breaks lactose down into galactose and glucose, simple sugars that can enter into glycolysis

• low levels of LacZ → convert lactose into allolactose ligand inducer to increase operon expression

• organization: lacZ, lacY, and lacA under the control of one promotor, lacI under the control of a separate promotor that produces LacI repressor that binds to lacOI and lacO

• scenario 1: no lactose- LacI repressor forms a tetramer that binds to lacOI and lacO and engineers a DNA loop that makes the lacZYA promotor inaccessible and thus prevents transcription

• scenario 2: only lactose- at low LacZ concentration, it produces allolactose inducer that binds to LacI to reduce its operator infinity and thus increase operon expression and since cAMP levels are higher without glucose present, cAMP binds to cAMP receptor protein (CRP) activator so it can recruit RNA Pol and increase its affinity for lacP and thus increase operon expression

• scenario 3: both lactose and glucose- IIB phosphorylates glucose instead of IIA so unphosphorylated IIA inhibits LacY and lactose cannot be imported and since cAMP levels are lower with glucose present, CRP does not bind to DNA and the operon is not expressed → catabolite repression (use up most energy-efficient carbon source first)

two-component system

• common regulator of transcription: environmental signal binds to sensor kinase → triggers autophosphorylation of sensor kinase → sensor kinase transfers its phosphate to a response regulator (can be an activator or a repressor) → phosphorylated response regulator binds DNA and either represses or activates the target genes → phosphatase removes the phosphate to inactivate it

• number of two-component systems reflects the lifestyle of a bacterial cell depending on how unpredictable its environment is: intracellular bacteria or endosymbionts like Wolbachia only have 1 or 2, while free-living bacteria like Nostoc can have 120

post-transcription and translation

• small RNA (sRNA)

  • regulates translation: inhibits translation by binding to and covering RBS and activates translation by uncovering RBS by binding to and unfolding hairpin

  • regulates stability of mRNA: promotes mRNA degradation by exposing RNase binding sites and prevents mRNA degradation by covering RNase cleavage site

transcription and translation

• riboswitch

  • transcriptional control: secondary RNA structure that when bound with a ligand undergoes a conformational change (antiterminator → terminator that blocks coding sequence) that terminates transcription

  • translational control: thiamine pyrophosphate (TPP) binds to RNA secondary structure to induce a conformational change into a tertiary structure that shifts the RBS over so it is inaccessible thus blocking translation

post-translation

• heat-shock response

  • at normal temperature, rpoH gene for RpoH sigma 32 is transcribed but mRNA secondary structures block its RBS and DnaK-DnaJ-GrpE chaperones shunt it to degradation

  • after a sudden temperature increase, Dnak-DnaJ-GrpE bind to denatured proteins to refold them and mRNA secondary structures melt, exposing the RBS so RpoH sigma 32 instantly accumulates and drives the expression of heat-shock genes (DnaJ, GrpE, DnaK, itself) → positive feedback

quorum sensing

• Vibrio fischeri: produce light via quorum sensing in Bobtail squid to hide their shadow from other creatures

  • positive feedback in gene regulation: LuxI synthesizes an acyl homoserine lactone autoinducer that diffuses into confined environment and accumulates until it reaches a threshold concentration and diffuses back into cell → binds with LuxR activator to activate lux operon to produce luciferase that makes light and LuxI (positive feedback)

• allows bacteria to exist as social organisms: adapt to biofilm, cause disease, confound competitors, and attack as a ‘wolf pack’

• not all bacteria are capable of quorum sensing

• different bacteria have different autoinducers and some have > 1 quorum sensing mechanism

regulon

a group of operons regulated by the same transcription factor despite being located at different parts of the bacterial chromosome