Gene Expression and Pathogenesis

Question 1

What is bacterial DNA packaged by?

What shape are bacterial genomes? How long is E Coli genome?

Answer 1

Supercoiling and histone-like proteins

Circular (4.64 million base pairs, Mbp)

Question 2

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?

Answer 2

~4500, ~4400

1000-2000

10-20 copies/cell (eg: repressor) to 200k copies/cell (eg: ribosomal protein) [differences due to transcription/translation regulation]

Question 3

How can we look at the proteome of a bacteria?

Answer 3

Separate them based on charge (isoelectric focussing - IEF) and then on weight (SDS-page)

Question 4

What is constitutive expression of genes?

At what levels could you regulate gene expression?

Answer 4

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

Question 5

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?

Answer 5

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

Question 6

What enzyme charges tRNA?

What is the ‘reading frame’?

Subunits of ribosome?

Sites in ribosome?

Answer 6

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!)

Question 7

How does the ribosome distinguish the start codon from other AUG codons in bacteria?

Answer 7

Shine Dalgarno sequence

Ribosome: UCCUCCACUAG

mRNA: AGGAGGX_4-7AUG

Question 8

What is a polysome?

Why does this interfere with rho-dependent transcription termination?

Answer 8

mRNA coated with ribosomes (occurs in prokaryotes)

Ribosomes stop rho protein from being able to reach RNAp

Question 9

What proteins fold other proteins?

What needs to happen for a protein to be directed to the appropriate part of the cell?

Answer 9

Chaperonins (chaperone proteins - usually barrel shaped)

Signal addition/cleavage

Question 10

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?

Answer 10

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)

Question 11

RNA Polymerase and sigma factor. Which would be the core enzyme? What about the holoenzyme?

Answer 11

Core = RNAp, holo = RNAp + sigma

Question 12

What two types of transcription termination are there in bacteria? How do they work?

Answer 12

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

Question 13

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?

Answer 13

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.

Question 14

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?

Answer 14

Operon

16S rRNA, tRNA, 23S rRNA, 5S rRna, tRNA

Question 15

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 ________ _______).

Answer 15

operator, dimers, major groove, consensus sequences, two-fold rotational, inverted repeats

Question 16

Name three common DNA binding motifs

Answer 16

Helix-turn-helix, zinc finger, leucine zipper

Question 17

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 __________ _____

Answer 17

Linkers

Regularly interspersed leucine residues

A zinc ion, two histidine residues, and two cysteine residues

Recognition helix

Question 18

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?

Answer 18

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.

Question 19

Lactose operon is switched on when lactose is present in the media. What happens at the molecular level?

How is maltose breakdown different?

Answer 19

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).

Question 20

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?

Answer 20

Proximal and distal regulation (it has to bend so the activator and the promoter are nearby each other)

Question 21

What is diauxic growth? What is this an example of? Why do organisms do this?

Answer 21

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

Question 22

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?

Answer 22

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)

Question 23

What process controls the expression of genes for amino acid and nucleotide synthesis?

Answer 23

Attenuation

Question 24

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 (?)]

Answer 24

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.

Question 25

What does the term ‘cis acting’ and ‘trans acting’ refer to in regulation?

Answer 25

Trans: far away (another molecule expressed from a different gene regulates the gene)

Cis: close by (elements binding to promoter regions that affect transcription)

Question 26

How do small untranslated RNAs regulate translation? What is this also known as?

What do ncRNAs regulate in Staph Aureus?

Answer 26

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)

Question 27

What is a riboswitch?

What is thiamine?

Answer 27

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]

Question 28

What is quorum sensing?

What tends to be the signal in gram neg/gram pos bacteria?

Answer 28

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

Question 29

What is a biofilm?

Answer 29

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)

Question 30

What group behaviours are affected by quorum sensing? [8]

Answer 30

Symbiosis, virulence, competence (ability to take DNA on board), conjugation (to take DNA from other bacteria), antibiotic production (by fungi), motility, sporulation, biofilm formation

Question 31

What is an example of quorum sensing symbiosis in fish?

Answer 31

Vibrio fischeri in fish that display bioluminescence (only occurs when bacteria are present in large enough numbers) - gives advantage to both species.

Question 32

What are two component systems (response regulators)?

What are these also known as? Why?

Answer 32

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

Question 33

Two component systems: what enzyme will switch off the system?

Answer 33

A phosphatase (often controlled by some other pathway, or just by timing, etc)

[NB: image probably won’t be in exam]

Question 34

What niche specific environmental conditions can affect microbial growth (or can push them to take action to create biofilms, etc)? [5]

Answer 34

pH, temperature, oxygen levels, micronutrient availability, host defence mechanisms

Question 35

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

Answer 35