Gene Expression and Pathogenesis Flashcards
What is bacterial DNA packaged by?
What shape are bacterial genomes? How long is E Coli genome?
Supercoiling and histone-like proteins
Circular (4.64 million base pairs, Mbp)
E coli has how many genes? How many potential proteins?
How many of these proteins are made in a growing cell?
Protein abundance - ranges from/to (per cell)? What causes this large range?
~4500, ~4400
1000-2000
10-20 copies/cell (eg: repressor) to 200k copies/cell (eg: ribosomal protein) [differences due to transcription/translation regulation]
How can we look at the proteome of a bacteria?
Separate them based on charge (isoelectric focussing - IEF) and then on weight (SDS-page)
What is constitutive expression of genes?
At what levels could you regulate gene expression?
Continual expression - housekeeping genes
At transcription (no mRNA), at translation (no protein product), at the level of protein activity (stop it from having assigned function), or let it work as intended
What is the start codon? What does it code for?
Termination codons?
What is an anticodon?
What would the anticodon for the initiation protein be?
AUG - (f)Met
UGA, UAA, UAG
Triplet on tRNA that binds to mRNA at ribosomes (relates to identity of amino acid that tRNA is charged for)
UAC
What enzyme charges tRNA?
What is the ‘reading frame’?
Subunits of ribosome?
Sites in ribosome?
Amino-acyl tRNA synthetases
The frame of three codons that are being read (-2/-1/+1/+2 = frameshift)
Large (50S), small (30S)
Aminoacyl (where tRNA docks), peptidyl (where bond is forged), exit (goodbye tRNA!)
How does the ribosome distinguish the start codon from other AUG codons in bacteria?
Shine Dalgarno sequence
Ribosome: UCCUCCACUAG
mRNA: AGGAGGX4-7AUG
What is a polysome?
Why does this interfere with rho-dependent transcription termination?
mRNA coated with ribosomes (occurs in prokaryotes)
Ribosomes stop rho protein from being able to reach RNAp
What proteins fold other proteins?
What needs to happen for a protein to be directed to the appropriate part of the cell?
Chaperonins (chaperone proteins - usually barrel shaped)
Signal addition/cleavage
What needs to bind to RNA polymerase (in bacteria) to allow them to transcribe? How does this allow for regulation?
What happens to this substrate once transcription has begun?
Where does the substrate/RNA polymerase bind to DNA?
Sigma factors (different ones expressed at different times - alpha70: housekeeping, alpha32: heat shock, alphaF: sporulation)
Sigma factor falls off
As promotor sequences (-35 consensus sequence, pribnow/-10/TATA box)
RNA Polymerase and sigma factor. Which would be the core enzyme? What about the holoenzyme?
Core = RNAp, holo = RNAp + sigma
What two types of transcription termination are there in bacteria? How do they work?
Rho-independent (most genes): loop of As and Us in mRNA destabilizes binding of RNAp and transcription ends
Rho-dependent (often seen in genes that are not translated - ribosomes, tRNA, snRNA, etc): rho protein binds to mRNA, makes its way along - when a hairpin causes RNAp to pause, it catches up, and causes disassociation
There is a premature stop codon, which causes a ribosome to fall away from mRNA that is still being transcribed. What can the rho protein do in this instance? What is this called?
How do you stop rho from prematurely terminating rRNA and tRNA?
Can then attach to mRNA, and stop further transcription - both the mutated gene and any downstream genes are not transcribed (called ‘polarity’)
Anti-terminator proteins - they bind to RNAp and allow read-through of terminators.
What do you call a collection of genes that is transcribed by a single promoter?
Stable RNA genes are clustered in E coli. Which ones?
Operon
16S rRNA, tRNA, 23S rRNA, 5S rRna, tRNA
Repressors and activators bind at the ________. They usually come in the form of ______ that fit the _____ ______ of DNA. At these points there are _________ __________ that demonstrate ___-____ __________ symmetry (also known as ________ _______).
operator, dimers, major groove, consensus sequences, two-fold rotational, inverted repeats
Name three common DNA binding motifs
Helix-turn-helix, zinc finger, leucine zipper
What is the string of proteins that connect the two helixes of a helix-turn-helix motif?
What are the two parts of the leucine zipper motif held together by?
What is a zinc finger (commonly) held together by?
All DNA-binding motifs seem to have a __________ _____
Linkers
Regularly interspersed leucine residues
A zinc ion, two histidine residues, and two cysteine residues
Recognition helix
Arginine can be synthesised; it is what kind of amino acid? What happens if it is provided in the media?
How is this likely to occur at the genetic level? What is the molecule that will cause this process to occur? What is it called?
Non-essential - the enzymes making it will shut down
The genes producing the enzymes that create it are on an operon (ArgC/B/H). The respressor for this operon is usually not attached; when arginine (the co-repressor) is present, it binds to the repressor, which undergoes a conformational change and can then bind to the operator. This stops transcription of the associated genes.
Lactose operon is switched on when lactose is present in the media. What happens at the molecular level?
How is maltose breakdown different?
Repressor is bound to the operator normally. When lactose is present, it is converted to allactose (the co-repressor), and then binds to the operator (which falls off the operator, thus allowing transcription to occur). NB: also positive regulation via an activator (low glucose = high cAMP = increased lactose breakdown)
There is no operator for the maltose operon. An inducer (maltose) binds to an activator (which allows RNAp to bind, and then transcription of associated genes can occur).
What is it called when a regulatory element is near the promoter? When it is far away? What happens to DNA in the latter case?
Proximal and distal regulation (it has to bend so the activator and the promoter are nearby each other)
What is diauxic growth? What is this an example of? Why do organisms do this?
Catabolite repression: A microbe has a preferred energy source. It will use this once until it is exhausted, and then switch on genes for alternative sources (on a diagram you see a growth phase, followed by a flatter line, followed by another growth phase). It is done because the catabolism of more complex sources takes more energy
How does the activator of the lac operon work? What is it called? What enzyme is involved in creating the inducer? Where is its binding site?
Activator: Catabolite Activator Protein (CAP) - binds upstream of promoter (pribnow/TATA box and -35). ATP -> cAMP catalysed by Adenylyl cyclase.
CAP will only bind to its binding site when cAMP is bound to it. cAMP is only present in higher concentration when glucose levels are low (as otherwise it would be getting converted to ATP)
What process controls the expression of genes for amino acid and nucleotide synthesis?
Attenuation
Attenuation
What is the name of the sequence in operons that senses the level of amino acid in the cell? What is it? Where in the operon is it?
So what then happens if the cell is starved of the particular amino acid? What is there is plenty? [NB: for tryptophan - lysine and serine slightly different (?)]
Leader peptide - it is a repeated series of the the particular amino acid. It is found after the promotor and the operator, and is translated into mRNA
Remember it’s bacteria, so ribosome binds to extending mRNA. Leader peptide cannot be translated due to low amino levels, so ribosome stalls - this allows for an anti-terminator loop to form in the mRNA still to be translated. This does not interfere with transcription of the genes for amino synthesis. If there is plenty, then more of the mRNA is translated - a terminator loop forms in the mRNA, which causes RNAp to fall off the DNA.
What does the term ‘cis acting’ and ‘trans acting’ refer to in regulation?
Trans: far away (another molecule expressed from a different gene regulates the gene)
Cis: close by (elements binding to promoter regions that affect transcription)
How do small untranslated RNAs regulate translation? What is this also known as?
What do ncRNAs regulate in Staph Aureus?
Regulatory (anti-sense) RNA binds to a single stranged mRNA - this makes it partially double stranded, and blocks the ribosome from translating it. [AKA RNA interference]
Virulence (biofilm, iron metabolism, exotoxin production, hydrolytic enzymes)
What is a riboswitch?
What is thiamine?
a riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA - the small molecule can often be a product of the enzyme that the mRNA would code for
Vitamin B1. [functions via riboswitch as above]
What is quorum sensing?
What tends to be the signal in gram neg/gram pos bacteria?
Bacteria produce signal molecule at constant rate. Concentration therefore increases with cell density, and certain genes are expressed when the signal has increased enough.
Neg: acyl homoserine lactone, pos: peptides
What is a biofilm?
Layer of difficult to shift matter (thick, glue-like glycoproteins) that develops through quorum sensing. Gives resistance to attack. Gets rid of things that aren’t needed (eg: flagella)
What group behaviours are affected by quorum sensing? [8]
Symbiosis, virulence, competence (ability to take DNA on board), conjugation (to take DNA from other bacteria), antibiotic production (by fungi), motility, sporulation, biofilm formation
What is an example of quorum sensing symbiosis in fish?
Vibrio fischeri in fish that display bioluminescence (only occurs when bacteria are present in large enough numbers) - gives advantage to both species.
What are two component systems (response regulators)?
What are these also known as? Why?
A system that allows bacteria to sense changes in environment and respond (often by controlling gene expression). Consists of a membrane localized histidine/sensor kinase (phosphate added to a histidine residue) and a cytoplasmic response regulator (often a transcription factor) to which this phosphate is transferred (to activate it)
Phospho-relay system: because kinase transfers phosphate to response regulator
Two component systems: what enzyme will switch off the system?
A phosphatase (often controlled by some other pathway, or just by timing, etc)
[NB: image probably won’t be in exam]
What niche specific environmental conditions can affect microbial growth (or can push them to take action to create biofilms, etc)? [5]
pH, temperature, oxygen levels, micronutrient availability, host defence mechanisms
Ways e Coli can be made PATHOGENIC. NB: just study it - appreciate how e Coli can take on different repertoires of genes
PAI = Pathogenicity Islands
