11114269
Description
Flashcards by Candice Young, updated more than 1 year ago
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Created by Candice Young
over 6 years ago
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| Question | Answer |
| What must cells control in order to best respond to their environment? | Control of gene expression and enzyme activity (post transcriptional regulation) help best for this |
| Where is the ribosome binding site relative to the start of transcription on a bacterial gene? | Ribosome binding site is AFTER the start of transcription, right before the start codon |
| RNAP | core RNA polymerase enzyme with subunits α2ββ´ω σ factor attaches to it --> helps polymerase bind to promoter region |
| What makes for a strong promoter? | A better match of the promoter sequence (-10 bp) with the consensus sequence (-35 bp) makes for this, and in turn leads to more transcription of the gene |
| What is typically needed for a weak promoter to turn on? | Extra proteins with transcriptional factors are needed to turn this on |
| Accessory transcription factors | role: will promote or repress transcription of specific genes how: form dimers and bind to direct/inverted repeats can be an activator or a repressor |
| repressors | bind to DNA and block transcription of downstream genes, |
| corepressors | End products of biosynthetic pathways can act as this (in the presence of a repressor) |
| Arginine corepressor | binds to Arginine repressor protein --> prevents RNAP from transcribing arginine synthesis operon |
| Inducers | specific enzyme substrates that block action of repressor proteins |
| Lactose inducer | lactose present --> binds to and inactivates repressor of lac operon --> lactose utilized and B-galactosidase is cleaved |
| Activator | are turned on in the presence of coactivators, promote transcription of a specific DNA sequence |
| Maltose activator | no transcription without maltose AND maltose activator; maltose present --> extra binding contacts added to RNAP, maltose coactivator binds and increases binding affinity of sequence with promoter --> Maltose operon transcribed |
| How to determine difference between inducer vs activator (since phenotypic results are the same) | 1) Stain promotor of operon in interest w/ GFP --> WT strain with this plasmid will only make GFP when target substrate is present 2) mutagenize strain w/ a transposon (tn) 3) replica plate mutants on mixture of substances (including target substrate), one in UV and one in room light 4) those that are gray in UV most likely have a tn that is blocking expression of GFP AKA has complete loss of function --> loss of function of an ACTIVATOR calls for this 5) make another replica plate of Tn mutants with glucose + substrate & glucose only, visualize both with UV 6) Colonies green on glucose only --> have Tn insertions that allow something to be on when it should be turned off --> indicate loss of function of a repressor |
| the basics of how to tell an inducer apart from a coactivator | "something is turned on when it should be turned off" --> mutated REPRESSOR "something is turned off when it should be on" --> mutated ACTIVATOR |
| Types of repressor-corepressor mutations | 1) point mutation in operator --> repressor can't bind 2) repressor itself can get knocked out 3) point mutation in repressor --> can't bind corepressor |
| types of repressor-inducer mutations | 1) repressor knockout 2) point mutation in operator so it can't bind repressor 3) point mutation in repressor --> can't bind inducer |
| types of activator-coactivator mutations | 1) point mutation in activator binding site 2) activator knockout 3) point mutation in activator protein so that it no longer binds coactivator |
| What is the point of specific regulatory mechanisms? | They control the ACTIVITY of a few enzymes and to control the SYNTHESIS of a few proteins |
| Global regulatory mechanisms | allow a bacteria to respond to complex environmental changes as well as adapt to major changes in their physiology --> much of this involves controlling which sigma factors are present and active |
| catabolite repression | allows bacteria to use most efficient carbon source first |
| Diauxic growth curve | a graph of growth rates of cells grown on a mixture with both glucose and lactose --> lactose is used only after glucose is gone, which increases production of B-galactosidase as a result --> lag in growth is visible during switch |
| Catabolite Activator Protein (CAP) | in absence of glucose, activates transcription of genes for alternative sugar utilization --> is an activator, cAMP is a coactivator |
| cAMP | coactivator that binds to CAP and allows it to bind to DNA and activate transcription If glucose levels decrease, cAMP levels increase |
| Specific vs global regulatory mechanisms | can be used in COMBINATION for example, lac repressor and CAP |
| regulation of the lac system | Lacl repressor binds to operon and blocks tx UNLESS inducer lactose binds to it |
| PTS system | if glucose present: Enz IIa inhibits uptake of alternative sugars by binding to their transporters if glucose absent: Enz IIa-P binds to adenylate cyclase, stimulates production of cAMP |
| What happens in the cell when glucose is PRESENT? | 1) Glucose enters rapidly + rapidly flies through glycolytic pathway --> PEP/pyruvate ratio low 2) Low phosphorylation of PTS proteins 2) Enzyme IIa inhibits alternative sugars |
| What happens in the cell when glucose is ABSENT? | 1) glucose enters slowly + slowly goes through glycolytic pathway --> PEP/pyruvate ratio high 2) High phosphorylation of PTS proteins 3) Enz IIa-P binds and stimulates adenylate cyclase |
| How do cells recover from heat shock? | Cells can do this by having genes with promoter sequences that bind to σ32 (RpoH) AND/OR by synthesizing chaperones and proteins to refold or degrade damaged proteins |
| σ32 in normal conditions | RpoH gene is transcribed with a stem loop at 5' end of mRNA --> transcription is inefficient --> DnaK chaperone binds to it and makes an inactive complex --> DnaK targets σ32 for degredation by FtsH protease |
| σ32 in heat shock conditions | RpoH gene is transcribed with a stem loop at 5' end of mRNA --> heat melts stem loop --> tx efficient --> σ32 is not inactivated by DnaK and instead interacts with RNAP core to promote tx of heat shock induced genes *meanwhile DnaK promotes refolding or degredation of denatured proteins* |
| How do cells finally recover from heat shock? | by using a homeostatic mechanism! Dnak is turned on by σ32 itself --> over time denatured proteins are refolded and there are enough free DnaK to rebind σ32 --> refolded proteins are released, σ32 degraded/inactivated |
| SOS response to DNA damage | 1) RecA binds to ssDNA that is damaged 2) Complex stimulates LexA autocleavage 3) Will be either partially repressed (continuing the cycle) or fully repressed, meaning SulA (inhibitor of cell division) and UmuCD (translesion DNA synthesis) proteins can be transcribed |
| LexA | represses certain genes by binding to SOS boxes (specific DNA sites), is cleaved WHEN DNA is damaged by UV light |
| How do cells reset after SOS repair? | SulA is depleted by proteolysis and degraded by Lon protease--> cell division starts again, LexA represses tx UmuCD is depleted by proteolysis and degraded by ClpXP protease --> no more mutagenetic replication, LexA represses tx |
| Stringent response | enables bacteria to survive nutrient deprivation! uncharge tRNAs accumulate during aa deprivation, when one enters A site of a ribosome it is sensed by RelA --> Rel A synthesizes ppGpp using GTP and ATP |
| ppGpp | an alarmone produced by RelA in the ribosome helps cell survive amino acid starvation |
| Functions of ppGpp | 1) inhibits chromosome replication by binding to DnaG (initiation & elongation) 2) slows transcription by binding to RNAP and preventing rRNA & ribosomes from being made --> reduced translation 3) favors alternative σ factors binding to RNAP besides σ70, allows stress σ factors to bind instead |
| two-component signal transduction | two Histidine kinases dimerize and cross phosphorylate --> phosphoryl group is transferred to response regulator --> these can activate/repress tx, turn on/off enzymes, bind to downstream protein in one particular state (phos or dephos) |
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