lecture 1 - genome organisation, nucleoid associated proteins and bacterial transcription

why is prokaryotic gene regulation important?

to how bacteria control and express their genes

what do bacteria need to do?

< they are free living organisms that need to adapt to changing environmental conditions to survive

how are bacteria different to eukaryotic cells?

< unlike stable environments like the microvascular endothelial cell lining in blood vessels, bacteria face varying conditions

what is an example of a bacteria which faces significant environmental changes?

< Campylobacter bacteria, which causes diarrhea after consuming undercooked chicken, experiences significant environmental changes moving from bird guts (at 42 degrees) to human intestines (at 37 degrees).

what does gene regulation allow?

allows bacteria to quickly switch genes on/off and adapt to environmental changes, ensuring survival

what do bacteria often inhabit?

< competitive environments, such as the diverse ecosystems of bird guts or human intestines
< these environments host thousands of bacterial species competing for resources and nutrition.

what does efficient gene regulation allow?

allows bacteria to maximize resource utilization and competitive advantage

what is an example of a bacteria operating in a competitive environment?

Campylobacter bacteria must efficiently digest specific sugars present in their environment to thrive without wasting resources

what features does gene regulation have?

< enables bacterial cells to rapidly adapt to changing conditions
< this flexibility is crucial for survival in dynamic environments where conditions can fluctuate rapidly
< bacteria must remain flexible and adaptable to ensure their survival in diverse and competitive ecosystems
< cells must switch genes on/off, express them, and translate them to respond effectively to environmental cue

what is the structure of most bacteria?

< have a single circular chromosome
< with densely coded genetic material minimising non functional junk dna

do bacteria have introns?

< bacteria typically have few introns, eliminating the need for a spliceosome
< instead, some bacteria have self-splicing or repeating sequences of non-coding DNA.

what does the majority of the bacterial genome encode for?

< proteins or functional RNAs

what is the intergenic distance in bacteria?

< short intergenic distances are common
< the median intergenic distance in E. coli being around 60-70 base pairs < this proximity facilitates the coordination of regulatory signals required for gene expression, including promoters and transcription factor binding sites

are operons prevalent in bacterial genomes?

< yes - multiple genes are arranged consecutively without intergenic spaces
< a single promoter drives the expression of polycistronic mRNA, which encodes open reading frames of multiple genes that are translated separately

how are crashes between polymerases minimised?

< transcriptional units are often orientated in the same direction as the chromosome's replication fork
< circular genomes typically replicate unidirectionally to avoid collisions

what are exceptions to the orientation?

< genomes may undergo bidirectional replication
< so the orientation of open reading frames may alternate with transcription units and replication units move in the same direction to prevent collision

what challenge do bacteria face and what is an example of this?

< fitting large DNA genome into very small cells
< e.g. e coli cell is about 2 microns long and contains a 4.6Mb genome, resulting in a tight packing problem

what should the genome fit into?

< in a circular form
< must fit into a space with a circumference of only 1.6mm inside the 2-micrometer long cell

what is the first key mechanism utilised by bacteria to address challenge of fitting large DNA genome into small cell?

< organisation by proteins
< the genome is highly organised and constrained by proteins into protein domains
< nucleoid-associated proteins (NAPs) bind and organise genomic DNA into independently folded domains

regarding the first key mechanism utilised by bacteria to address space challenge, what do electron micrographs show?

< electron micrographs of gently lysed E.coli cells reveal distinct loops of DNA, each constrained by NAPs
< about 400 independent domains exist within the DNA molecule of E.coli

what is the second key mechanism utilised by bacteria to address challenge of fitting large DNA genome into small cell?

< DNA supercoiling
< this further compacts DNA
< each loop of DNA is independently coiled, allowing for further compression or relaxation as needed

what does the highly organized and constrained genome still needs to be accessible to?

DNA and RNA polymerases for transcription and replication

how is the organisation of the genome highly dynamic?

DNA coiled in multiple domains (~400), each capable of independent compression or relaxation

what are NAPs?

< nucleoid associated proteins
< play a crucial role in binding to DNA and assisting in its compaction to fit inside a bacterial cell

what NAPs do E coli have?

< most bacterial species produce multiple different NAPs, with E. coli, a standard model organism, having at least 6 NAPs:
< H-NS, Fis, IHF, HU, Dps, and CbpA
< each of these proteins has slightly different functions, and the activity of all of them is required for efficient cell functioning

what is H-NS?

< proficient at organising DNA and can form dimers
< it binds to DNA and interacts with itself
< facilitating the formation of loops of DNA by bringing together segments through protein-protein interactions
< can bridge adjacent segments of DNA, bringing distant parts of the genome into proximity

what does Fis and IHF do?

< proteins which combine relatively non-specifically with DNA, though they have preferential sequences
< they induce severe bends in the DNA aiding in its compaction into a smaller size

what do Dps and VbpA do?

< these proteins bind to and protect DNA from damage during times of stress
< they are expressed when the bacterium faces nutrient depletion, chemical stress, or environmental challenges.

what is HU?

< the most conserved NAP
< can condense DNA into a fibre like structure
< it resembles histone proteins in eukaryotes and organises itself into a fibre, wrapping DNA around the protein

Dps and CbpA in stationary phase?

< these are expressed during the stationary phase of bacterial growth
< they condense DNA to protect it from damage, forming tight knots to shield the DNA from stress-induced damage.

why do bacteria need supercoiling?

< to further compact DNA loops within the nucleoid

in a relaxed circle of DNA, how many base pairs are there per turn?

< approx 10

what is positive supercoiling?

< overtwisting the DNA helix leads to the spontaneous formation of a positive supercoil, introducing strain and energy into the helix
< this positive supercoil forms in a clockwise direction

what is negative supercoiling?

< under twisting the helix results in the formation of a negative supercoil which occurs in a counter clockwise direction
< negative supercoiling creates tighter knots, further compacting the DNA until it occupies the smallest possible space

how is supercoiling a dynamic process?

< within the independent loops constrained by proteins, DNA can be compacted into smaller knots
< this process is dynamic as DNA must be unravelled for replication and then recoiled afterwards
< the large genome constantly undergoes conformational changes to facilitate various processes

what are the functional implications of supercoiling?

< compacts DNA and adds/removes energy that can be utilised for transcription

what are the functional implications of positive supercoiling?

makes DNA more tightly coiled, hindering transcription or replication by making the DNA strands physically harder to pull apart

what are the functional implications of negative supercoiling?

reduces the energy threshold required to separate DNA strands, making transcription or replication easier within that region

how can bacteria modulate supercoiling?

to provide targeted energy to different parts of the genome, thereby regulating transcription or replication processes as needed

how is supercoiling of DNA controlled in bacteria?

< regulated by topoisomerases - enzymes that affect the topology of DNA altering its direction and mirror images

what are the types of topoisomerases in E.coli?

< E coli possesses four topoisomerases (I-IV)
< topoisomerase II, also known as gyrase, being the only enzyme capable of introducing negative supercoils
< the other three topoisomerases can relax supercoils or, under very limited conditions, occasionally introduce positive supercoils.

how is negatively supercoiled DNA dominant in bacteria?

< gyrase activity, which introduces negative supercoils, dominates this process, while the other topoisomerases (I, III, and IV) relax these negative supercoils as needed

what is the gyrase mechanism?

< gyrase is composed of two proteins: GyrB and GyrA which forms a heterodimer complex
< GyrB binds to DNA relatively nonspecifically
< GyrA induces a double-strand break in the DNA, remaining covalently bound to each end.
< GyrA is also an ATPase, hydrolyzing ATP, inducing a conformational change that passes the intact strand through the break.
< GyrB then religates the break.

what is the role of quinolone antibiotics?

< quinolone antibiotics like ciprofloxacin target gyrase by binding to and stabilizing this covalent complex, preventing the religation of DNA
< by inhibiting gyrase, quinolone antibiotics indirectly introduce a double-strand break into DNA, making them highly effective against bacterial infections

what is the emergence of resistance?

< there are many bacteria resistant to quinolone antibiotics due to small mutations in gyrase enzymes that prevent drug binding.
< these resistant bacteria pose significant challenges in antibiotic treatment.

how is supercoiling balanced in bacterial cells?

< by the activities of three main topoisomerases, each contributing to the regulation of DNA topology

how does Topoisomerase I function?

< by introducing a single-strand break in the DNA.
< when gyrase introduces a double-stranded break to relieve supercoiling, topoisomerase I capitalizes by passing one strand of DNA through the break, thereby relaxing negative supercoils.
< this process is repeated iteratively until a desired level of supercoiling is achieved.

are the activities of topoisomerases uniform across the entire genome?

no but they occur at individual domains of DNA

what do topoisomeresases II and III do?

< transiently relax individual DNA loops for transcription and replication
< maintain genomic stability and ensuring the proper functioning of DNA-related processes.

what do bacterial promotors consist of?

specific sequences upstream of the transcription start site (+1) and downstream of the gene to be transcribed

what does the promotor sequence typically include?

< a -35 sequence (TGTTGACA) and a -10 sequence (TATAAT)
< these numbers indicate their position relative to the transcription start site, with the -35 sequence being further upstream than the -10 sequence
< the +1 site marks where transcription initiates.

how many base pairs are there in ideal promotors?

there are typically 17 base pairs between the -35 and -10 sequences

what is the SD sequence and where is it located?

< for protein-coding transcripts, there is often a Shine-Dalgarno sequence (SD sequence)
< typically AGGAGG (although variations are tolerated)
< located shortly before the start codon (usually ATG) that marks the beginning of the protein-coding sequence.

how long is the gene that is transcribed?

< can vary in length, ranging from hundreds to thousands of base pairs
< after initiation RNA polymerase will continue transcribing until it reaches a terminator sequence.

what does the terminator sequence signal?

the end of transcription, causing RNA polymerase to dissociate from the DNA

what happens within the transcribed mRNA?

< the Shine-Dalgarno sequence facilitates the binding of the ribosome to the mRNA
< the ribosome then scans for the start codon (ATG) and initiates translation, synthesizing a protein until it reaches the stop codon (such as TAA).

what is the core RNA polymerase composed of?

< several subunits:
Two alpha subunits (α)
One beta subunit (β)
One beta prime subunit (β')
One omega subunit (ω)

where is this core polymerase complex present?

< in an inactive state
< floating within the cytoplasm of the cell

what does the core polymerase require?

< in order to bind to DNA and recognize a promoter sequence, the core polymerase requires the assistance of a sigma factor.

what is the function of sigma factors?

< play a crucial role in transcription initiation by forming an interface with DNA and recognizing specific promoter sequences.

what happens when a sigma factor binds to the core RNA polymerase?

< if forms a holoenzyme complex capable of initiating transcription

what does the sigma factor allows the RNA polymerase complex to do?

< The sigma factor allows the RNA polymerase complex to recognize and bind to the promoter region of DNA, facilitating the transcription of genes downstream of the promoter.

what does transcription initiation begin with?

the assembly of the RNA polymerase holoenzyme, which consists of the RNA polymerase core enzyme along with a sigma factor

what does the holoenzyme scan?

along the DNA after binding, specifically seeking promoter sequences

what happens when the holoenzyme encounters a suitable promoter?

< when it encounters a suitable promoter with consensus sequences at positions -35 and -10 relative to the transcription start site, the holoenzyme forms a closed complex with the DNA

what does the closed complex mark?

< the binding of RNA polymerase to the promoter, where it binds non-specifically and scans until the sigma factor recognizes a suitable promoter sequence, forming the closed complex

what happens after the closed complex is formed?

the RNA polymerase unwinds the DNA at the promoter region, forming an open complex

what happens in the open complex?

the RNA polymerase is positioned on a single strand of DNA, having unwound the helix, and is ready to initiate transcription in the direction it faces

what happens after initiation?

the sigma factor may dissociate from the RNA polymerase, allowing transcription to proceed

what is the core process of sigma factors?

< other sigma factors may exhibit slightly different mechanisms, but the core process involves the recognition of promoter sequences, formation of closed and open complexes, and initiation of transcription

how often does this process occur within the cell?

< continuously within the cell, with RNA polymerases directed to various promoters by sigma factors, ensuring the transcription of numerous genes to meet the cell's needs.

in bacteria how does transcription termination primarily occur?

< through two mechanisms:
< Rho-dependent and Rho-independent termination

what happens in rho-dependent termination?

< Rho is an mRNA-binding protein that recognizes a specific sequence following the gene being transcribed.
< as the RNA polymerase transcribes the gene, it includes the Rho recognition sequence in the mRNA.
< Rho binds to this sequence on the mRNA, trailing behind the polymerase.
< Rho catches up to the RNA polymerase, and upon contact, the polymerase stops and dissociates from the DNA, thereby terminating transcription.

what happens in Rho-independent termination

< Rho-independent termination relies solely on mRNA structure and does not require any additional proteins.
< A palindromic sequence in the mRNA forms a double-stranded GC-rich stem-loop structure.
< The RNA molecule can fold back on itself, with numerous GC residues in the palindromic sequence facilitating base pairing.
< this results in the formation of stable terminator stem-loop structures.
< as the mRNA is being transcribed, the terminator loop forms, and its presence is sensed by the RNA polymerase
< the contact between the terminator stem-loop and the polymerase leads to transcription termination.

in summary what does Rho-dependant termination involve?

the action of the protein Rho, which binds to a specific sequence in the mRNA and halts transcription when it catches up to the RNA polymerase.

in summary what does Rho-independent termination rely on?

intrinsic RNA structure, where the formation of stable stem-loop structures in the mRNA signals the RNA polymerase to terminate transcription spontaneously.

what is a characteristic of the fundamental processes of transcription and translation?

< near universal for all protein-coding genes
< but it's essential to understand that every step in these processes is regulated in every bacterial cell continuously

how do bacteria exert meticulous control over gene expression?

by regulating the rate of transcription initiation, suppressing termination, and controlling the rate of translation

what does the tight regulation allow?

bacteria to quickly adapt and alter their gene expression profiles in response to changing environmental conditions