Block B - Microbial Gene Regulation

Question 1

Which one is more stable, eukaryotic mRNA or bacterial mRNA?

Answer 1

eukaryotic mRNA

Question 2

What is the shine-dalgarno sequence and what process is it involved in? (3 marks)

Answer 2

-its a ribosomal binding site found in the mRNA of prokaryotes like bacteria. its rich in purines.
-its critical in translation initiation since the site binds to the 16S rRNA of the 30S ribosome subunit.
-The binding helps align the ribosome with the start codon on the mRNA, ensuring translation begins at the correct site.

Question 3

What is an operon? (2 marks)

Answer 3

its a functional unit of DNA consisting of a group of genes under the control of a single promotor. these are transcribed together and usually encode proteins with related functions, often involved in shared metabolic pathways.

Question 4

What is the importance of operons? (3 marks)

Answer 4

-by grouping genes under a single promotor, operons ensure coordinated expression of genes that work together, saving energy and resources
-operons allow bacteria to adapt quickly to changes in the environment by turning gene sets on and off in response to external changes
-control of operon control expression of entire function

Question 5

in bacteria, transcription is catalysed by?

Answer 5

RNA polymerase

Question 6

Describe the structure of RNA polymerase?

Answer 6

its a multi-subunit enzyme, composed of core enzyme which is made up of five subunits; α (alpha), β (beta), β’ (beta prime), and ω (omega). also has a sigma factor.

Question 7

What is the role of a sigma factor in RNA polymerase?

Answer 7

a detachable subunit that helps the core enzyme recognize and bind to the promotor regions of DNA.

Question 8

What are the 3 main stages of transcription?

Answer 8

initiation, elongation and termination

Question 9

What does the sigma factor interact with to form holoenzyme at the beginning of transcription initiation?

Answer 9

RNAP

Question 10

What is a closed complex in terms of transcription?

Answer 10

when the RNA polymerase binds to the promotor, but the DNA remains double stranded

Question 11

What is a open complex in terms of transcription?

Answer 11

when DNA unwinds aka DNA duplex opening, forming the transcription bubble

Question 12

In bacteria, where does transcription usually begin and why? (3 marks)

Answer 12

usually begins near the pribnow box in bacterial promotors, where the DNA is rich is adenine and thymine, which have fewer hydrogen bonds and are easier to seperate

Question 13

What happens in trancription elongation? (1 mark)

Answer 13

RNA polymerase synthesizes an RNA molecule by adding ribonucleotides which are complementry to the DNA template strand. it maintains the transcription bubble until transcription has been terminated

Question 14

Name some elongation factors and its affects (3 marks)

Answer 14

Pause sites-certain DNA sequences cause the RNAP to pause, which can regulate elongation speed
Proteins such as NusA, NusG and DksA associate with RNAP can reduce the likelihood of pauses by modulating RNAP interactions with DNA
Supercoiling also influences elongation, positive supercoiling makes the DNA duplex tighter and harder, while negative supercoiling loosens the DNA duplex

Question 15

Tell me about intrinsic termination/rho independant termination? (4 marks)

Answer 15

-is the mechanism of prokaryotic RNA synthesis termination without additional proteins (rho protein)
-in GC rich region, hairpin forms due to complementry base pairing
-hairpin destabilises DNA-RNA hybrid in transcription bubble
-downstream of the hairpin, the poly U tail is rich with AU bases, these are weak bonds and allow RNA to dissociate from DNA, stopping transcription

Question 16

Tell me about Rho-dependant termination (5 marks)

Answer 16

it requires the rho protein, which binds to a rho utilisation site in the mRNA. it catches up to RNAP and when the RNAP pauses at a termination site, rho disrupts the RNA-DNA hybrid using its helicase activity, causing RNAP to release the transcript to release and dissociate

Question 17

What are transcription factors?

Answer 17

these are proteins that regulate the transcription of specific genes by binding to nearby DNA sequences. they are essential for controlling the rate of transcription by activating or repressing gene expression.

Question 18

Most activator proteins only bind to DNA unless their _____ is present

Answer 18

inducer

Question 19

Name an example of alternative gene regulatory strategies (4 marks)

Answer 19

DNA methylation of the promotor of the CDKN2A gene (which encodes the tumour suppressor p16) can silence its expression in many cancers, enabling uncontrolled cell proliferation

Question 20

Explain how PrfA thermometer regulates genes in Listeria monocytogenes

Answer 20

-at temperatures below 30°C, the RBS is inaccessible due to secondary structures
-At 37°C (host conditions), the structure unfolds, initiating translation of PrfA, which activates virulence factors for infection

Question 21

For Listeria monocytogenes, why are virulence factors only expressed at 37°C? (3 marks)

Answer 21

this is because this temperature is the typical core body temp of mammals. this regulation ensures that virulence factors, which are energetically costly to produce, are only expressed when the pathogen is in a host and can cause infection

Question 22

Tell me about transcriptional attenuation in trp operon?

Answer 22

controls gene expression based on the availability of tryptophan in the cell. if trp is high, the ribosome translates the leader peptide, allowing it to move past the two tryptophan codons. this exposes regions 3 and 4 which form the terminator hairpin, halting transcription of the operon. if trp is low, ribosomes stall at the tryptophan codons because tryptophan-charged tRNA is scarce. this prevents the 3:4 hairpin from forming and promotes the 2:3 anti-terminator hairpin allowing transcription to continue

Question 23

Tell me about riboswitches (5 marks)

Answer 23

These are regulatory RNA elements, these directly sense small molecules or ions to regulate gene expression. they act as molecular switches, modulating processes based on the presence of specific ligands. they do not require proteins for their regulatory function. for example the thiamine pyrophosphate (TPP) riboswitch in bacillus subtilis stabilizes a terminator hairpin, halting transcription of the thiamine biosynthesis genes.

Question 24

Why do we need a high diversity of riboswitches?

Answer 24

because riboswitches are specific to a particular ligand, a high diversity ensures a large variety of metabolites can be targeted and regulated

Question 25

How can sRNA modulate DNA? (3 marks)

Answer 25

sRNAs can influence transcription by interacting with the transcription machinery or modifying chromatin structure. sRNAs can hybridize with complemenary sequences in mRNA, leading to translation repression by blocking ribosome binding sites (RBS). It can also lead to translation activation by unmasking RBS by destabilizing inhibitory structures.

Question 26

Give an example of a bacteria using sRNA to modulate DNA? (3 marks)

Answer 26

RyhB in E. coli, regulates iron metabolism genes. when under iron starvation, RyhB represses non-essential iron-using proteins by pairing with their mRNAs, these RhyB bound mRNAs are targeted by the enzyme RNase E causing degradation. this is facilitated by Hfq which stabilises RyhB and enhances its binding with target mRNAs.

Question 27

Tell me about how regulation by RNA-binding proteins (RBPs) regulates DNA.

Answer 27

RNA binding proteins indirectly regulates DNA by interacting with RNA transcripts. They control their stability, translation, and localization. RBPs can bind to specific sequences in the mRNA, either stabilizing of degrading the mRNA which determines how much of a protein is produced from a gene.

Question 28

Give me an example of RNA binding protein (RBP) regulation in bacteria? (3 marks)

Answer 28

the CsrA protein in E. coli. in nutrient abundance, CsrA binds to the RBS, CsrA represses genes for glycogen biosynthesis (glgCAP) by blocking their translation. during starvation, sequestered CsrA allow the translation of glgCAP, promoting glycogen storage.

Question 29

How does anti-termination regulate DNA? (3 marks)

Answer 29

anti-termination interfere with the normal termination of transcription. it prevents RNA polymerase from pausing and disengaging at termination sites. instead RNAP bypasses termination sites and continues transcribing downstream DNA sequences.

Question 30

Give me an example of anti-termination in bacteria? (2 marks)

Answer 30

antitermination in E. coli involves the λ (lambda) phage and its N protein. NusA and NusG are essential cofactors for the activity of the N protein that allows transcription to continue past termination sites in the early genes of the lambda phage genome.

Question 31

What is the stringent response?

Answer 31

its a global regulatory mechanism used to adapt to nutrient stress, particularly amino acid starvation. when amino acid levels drop, uncharged tRNAs accumulate in the cell. these uncharged tRNAs bind to the ribosomes A site, stalling translation. stalled ribosomes activate the enzyme RelA, which synthesizes an alarmone called (p)ppGpp. RelA and SpotT produce (p)ppGpp. (p)ppGpp acts as a global signalling molecule that reprograms transcription and other cellular processes. (p)ppGpp binds to RNAP and reduces its affinity for promotors of rRNA and tRNA genes, resulting in slowed ribosome production.

Question 32

Give me an example of stringent response in bacteria? (1 mark)

Answer 32

during amino acid starvation, e. coli synthesizes (p)ppGpp, repressing rRNA and tRNA transcription while activating amino acid biosynthesis operons.

Question 33

Explain how phase variation regulates DNA (2 marks)

Answer 33

phase variation is commonly DNA inversions, which involves the flipping a promotor or another regulatory region.
this can result in the expression or silencing of a particular gene.

Question 34

Give an example of how bacteria use phase variation to regulate DNA. (2 marks)

Answer 34

salmonella enterica controls flagellar phase variation by flipping a DNA segment that alternates between expressing two flagellin genes (fliC and fljB). This allows the bacterium to produce different flagellar proteins and evade immune detection.

Question 35

Why do bacterial activator proteins require their specific inducers to bind to DNA effectively

Answer 35

because the inducer causes a conformational change that enhances the protein’s ability to recognize and bind its target DNA sequences. Without the inducer, these proteins generally have a low affinity for DNA and cannot initiate transcription.

Question 36

Briefly how does a ribothermometer work? What formation is necessary? (3 marks)

Answer 36

1 a ribothermometer changes its conformation based on temperature. the formation of the RNA into a hairpin is necessary.
2 at low temperatures, the hairpin structure is stable, preventing downstream processes like translation.
3 As the temperature increases, the RNA can denature which causes a conformational change. The structures unfolds, exposing a region where the ribosome can bind for translationt