BIO 125 | 2.1 Transcriptional Regulation

T/F: Microbes regulate gene expression at three different levels

TRUE

3 gene regulatory mechanisms utilized by microbes

1. Transcriptional regulation

2. Translation regulation

3. Regulation of protein activity

Explain

Microbes regulate gene expression at 3 levels

• Transcriptional regulation

  • Control synthesis of RNA from DNA

  • DNA with promoter region (-35, -10, +1), RBS, structural gene that will be transcribed

  • Cellular signals

    • Activators promote transcription by helping RNA polymerase bind to promoter region

    • Repressors block transcription by preventing RNA polymerase from binding

  • If transcription is activated, mRNA is produced. If repressed, transcription is blocked.

• Translation regulation

  • Occurs at initiation of translation

  • Cellular signals

    • Riboswitches: regulatory segments of mRNA that bind to specific ligands, changing their conformation and hiding RBS from ribosome in hairpin structures.

      • In the absence of ligands, riboswitches will remain in open conformation and won’t be able to block translation.

    • sRNAs: noncoding RNAs regulate translation through direct base-pairing interactions with mRNA

      • sRNAs sometimes bind to mRNA, overlapping with RBS and thus preventing ribosome from physically binding to it

      • sRNA could also promote translation if it binds to mRNA in such a way that disrupts inhibitory structures like hairpin, exposing RBS to ribosome.

• Regulation of protein activity

  • Covalent modification, e.g., phosphorylation could activate or deactivate protein’s function

  • Protein-protein interactions: proteins forming complexes that are only functional when certain conditions are met

  • Degradation: Proteins no longer needed are marked for degradation to prevent accumulation

  • Feedback inhibition: Final product of pathway inhibits earlier steps of pathway to prevent overproduction of products and maintain homeostasis

_ interacts with major groove DNA to control transcription

DNA-binding proteins (DNABPs)

Part of DNA-binding protein that fits into major groove of DNA and interacts with base pairs

Domain

Parts of protein with specific structures and functions

Domain

Property of DNABPs having 2 identical subunits with various domains

Homodimerism

Where do DNABPs bind in DNA

They bind to inverted repeats in major groove DNA, which is within promoter region

Specific sequences that are identical when read in opposite directions in 2 strains; binding site for DNABPs

Inverted repeats

Why DNABPs bind to major groove DNA

Provides more access to nucleotide bases, making it a more suitable site for protein binding

Explain how DNABPs control transcription

• DNABPs recognize specific sequences, often the inverted repeats, in the major groove DNA (which provides more access to nucleotide bases than minor groove).

• DNA-binding domain of protein will then interact with base-pairs within recognized sequence.

• The binding of DNABPs to these specific sites can influence whether a gene is turned on or off.

Specific regions of protein with specific folding structure and function

Domains

Typical site of binding, usually at inverted repeats

Major groove

Various DNABPs interact with DNA in _ and control transcription

promoter region

Specificity of DNABPs depend on

interactions between several DNABP amino acids and DNA bases

Explain

• Asparagine 51 (Asn51) of DNABP interacts with Thymine-Adenine (TA) base pair on the DNA by forming hydrogen bonds with the N & O atoms of the bases.

• DNABPs can read and recognize specific sequences based on the arrangement of hydrogen bond donors and acceptors in DNA.

T/F: Each DNABP amino acid interacts with a base through hydrogen bonds, hydrophobic interactions, or other forms of molecular recognition.

TRUE

T/F: DNABP specificity arises from their AA side chains fitting into major grooves of DNA and forming specific interactions with the DNA bases

TRUE

T/F: Many DNABPs interact with DNA minor groove than major groove

FALSE

DNABPs bind more often on major groove since it’s wider and the bases are more exposed, allowing for more specific interactions. Meanwhile, minor grooves are narrower and some AA are still intact, making it less accessible to DNABPs.

Common structural motifs for DNABPs

• Helix turn helix proteins

• Helix loop helix

• Homeodomain

• Zinc finger

• Leucine zipper

• Beta sheet recognition proteins

T/F: Helix-based motifs (HTH, HLH, Leucine zipper) primarily bind the major groove DNA

TRUE

Why is helix turn helix the most common structural motif for DNABPs?

• Its recognition helix fits precisely into the major groove of DNA, allowing for specific base pair interactions.

• Its stable structure, with 2 helices connected by a short turn, ensures proper orientation for effective binding.

• Additionally, the simplicity and versatility of its structure make it adaptable across a wide range of DNABPs, from repressors to activators.

First helix, responsible for recognizing and binding to specific DNA sequences

Recognition helix

Second helix maintaining overall structure of motif

Stabilizing helix

Short loop connecting 2 helices in HTH motif

Turn

How to increase binding selectivity of DNABP

Dimerizing DNABPs allow for more selective binding to sequences

T/F: Cis-regulatory sequences are regions of noncoding DNA that regulates transcription of nearby genes by encoding the proteins themselves.

FALSE

Noncoding. Instead they serve as binding sites for proteins (e.g., transcription factors), which can enhance or repress transcription of associated genes.

Cis-regulatory sequences vs. Trans-regulatory

• Cis-regulatory sequences are located on the same DNA molecule as the gene they regulate.

• Trans-regulatory sequences are factors coming from other parts of genome and can affect gene from a distance, acting on cis-regulatory elements.

How does dimerizing a DNABP increase binding selectivity?

• Dimerizing DNABPs increase their binding selectivity because it extends the length CRS necessary for binding.

• With 2 recognition sites in play, the protein now has to recognize a longer and more specific sequence, making it less likely to bind to non-target DNA regions.

• This higher specificity will ensure that only sequences matching the full binding site are targeted, thus reducing off-target interactions.

Explain noncooperative vs. cooperative binding

• Noncooperative binding is when DNABPs already exist as dimers before binding to CRS.

  • Noncooperative bc each binding event is independent of others.

  • Exponential curve because binding is proportional to the DNABP concentration, meaning that as DNABP concentration increases, more dimers bind to CRS, leading to gradual, continuous increase of DNA occupancy.

• Cooperative binding is when DNABPs exist as monomers and only dimerize upon binding to CRS.

  • Cooperative bc the binding of 1 monomer to CRS increases likelihood of second monomer binding.

  • All-or-none phenomenon leading to sigmoidal curve because cooperativity feature creates a threshold effect, where at low concentrations, binding will be slow because monomers need to form dimers.

  • But, once critical concentration is reached, binding will be rapid, leading to sharp increase in DNA occupancy and creating sigmoidal curve.

T/F: All activators are transcription factors

FALSE

Not all activators are transcription factors. Activators are molecules or proteins that enhance gene expression, but they don't have to directly bind to DNA. Transcription factors, on the other hand, are proteins that regulate gene expression by binding to specific DNA sequences. Some activators work indirectly, influencing transcription factors or other proteins involved in gene regulation.

Proteins that control the rate of gene transcription by binding to specific DNA sequences

Transcription factors

Proteins increasing transcription rate; bind to activator binding sites

Activator

Proteins decreasing transcription rate; bind to operator region

Repressor

Phenomenon where binding of effector molecule changes conformation of transcription factor

Allosterism

Molecules binding to TF, changing its conformation and switching transcription on/off

Effector

Effector that switches transcription on

Inducer

Effector that switches transcription off

Corepressor

Transcription factors can enhance transcription rates through _ and decrease transcription rates through _

• Induction, derepression

• Repression

T/F: Activities of activator and repressor proteins are complex forms of regulation that repress or induce protein production.

FALSE

Simple forms of regulation

Transcription rate control works based on availability of final product

Repression

Explain why repression usually occurs in anabolic enzymes

• Because anabolic enzymes are enzymes involved in biosynthetic pathways that build complex molecules and repression ensures that unnecessary enzyme production stops once enough of the end product is produced to conserve cellular resources.

• In the case of arginine biosynthesis, once the end product (arginine) accumulates, it will act as corepressor and bind to ArgR repressor, causing it to bind to the operator region and block further transcription of biosynthetic enzymes.

• This prevents further synthesis of arginine when sufficient levels are already present.

What acts as corepressor to turn enzymes off in enzyme repression

Final product of biosynthetic pathway

Objective of repression

To ensure that unnecessary enzyme production stops once enough of the end product is present to conserve cellular resources.

Is arginine operon repressible or inducible

Repressible

Is lac operon inducible or repressible

Inducible

Transcription rate control where lack of substrate or presence of inducer turn enzymes on

Induction

T/F: Induction typically occurs in catabolic enzymes

TRUE

When needed substrate is present (e.g., lactose), it will trigger enzyme production

Process where repressor protein is inactivated by an inducer to allow genes to be transcribed

Derepression

When is the only time lac operon is turned on?

When bacteria does not have glucose and only have lactose

LAC OPERON ON/OFF?

• +G, +L =

• +G, -L =

• -G, -L =

• -G, +L =

• Off

• Off

• Off

• On

Explain induction / depression of lac operon

• Lac operon will only be turned when bacterium don’t have glucose but instead have lactose.

• In the absence of lactose, the LacI repressor will remain bound to lac operator region, preventing RNA polymerase from transcribing genes necessary for lactose metabolism.

• In the presence of lactose, allolactose will act as inducer by binding to LacI repressor, causing it to detach from lac operator region and subsequently allowing RNA polymerase to transcribe genes for lactose metabolism.

• This ensures that enzymes for metabolism of certain substrates will only be produced when substrates are actually present.

Aim of induction

To ensure that enzymes necessary for metabolism of certain substrates will only be produced when those substrates are actually present to conserve cellular resources

Form of lactose acting as inducers of LacI repressor

Allolactose

T/F: Activators are not always needed by RNA polymerase

TRUE

If promoter is sufficiently strong and RNA polymerase can easily recognize and bind to it, transcription can proceed at baseline level.

T/F: In the presence of activators, basal transcription rate is increased. In presence of repressors, basal transcription rate is decreased.

TRUE

T/F: Activator proteins can only bind to activator binding sites close to promoter.

FALSE

Activator proteins can bind to ABS both close to and far away from the promoter.

T/F: In many cases, activator protein binding is necessary because some promoters have poor matching to RNA polymerase.

TRUE

Activator protein function

To help RNA polymerase recognize the promoter and begin transcription

T/F: Looping may be necessary to activate RNA polymerases using some activators

TRUE

How is an activator binding site being hundreds of base pairs away from promoter region remedied

• The DNA must loop to bring the activator binding site in close proximity to the promoter region, such that activator protein will be able to physically interact with RNA polymerase, boosting transcription rate.

Use of inducers to regulate the activators controlling RNA polymerase

Positive control

Explain this case of positive control

• In the absence of maltose (inducer of malEFG operon),

  • neither the MalT activator protein and RNA polymerase binds to DNA

• In the presence of maltose,

  • it will bind to the MalT activator protein, subsequently causing it to bind the activator binding site.

  • Binding of activator protein to ABS will allow recruitment of RNA polymerase to promoter region and begin transcription.

A group of operons under the control of a single regulatory protein

Regulon

Operon vs. Regulon

• Operon

  • Genes grouped together physically in 1 chromosome, under the control of 1 promoter / regulatory element

  • e.g., LacI repressor can only bind to lac operon and regulate transcription of lactose metabolism genes located in that one position.

• Regulon

  • Multiple operons scattered throughout genome but regulated by 1 regulatory protein

  • e.g., MalT regulatory protein can bind to different operons within the regulon, allowing genes for maltose utilization to be expressed in various locations of the genomes.

T/F: The genes and operons required for maltose utilization (mal) are dispersed throughout the E. coli genome and regulated by the same maltose regulatory protein (MalT)

TRUE

Advantages and disadvantages of regulon

• Advantages

  • Coordinated gene expression across dispersed locations

    • Useful when the cell needs to respond quickly to a specific environmental signal, such as the presence of a nutrient or stress factor.

    • Also, in situations where multiple genes in different locations need to be turned on (e.g., during nutrient scarcity or in stress conditions), a single regulatory protein can trigger their expression simultaneously, making the response faster and more efficient.

  • Optimization of Resource Usage

    • By coordinating the expression of multiple genes under one regulatory signal, the cell can ensure that related genes are expressed only when needed, thus conserving energy and resources for other processes.

• Disadvantages

  • Global Disruption in Case of Mutation

    • Since a single regulatory protein controls multiple operons, a mutation in the regulatory gene or its protein can lead to widespread disruption across the entire regulon. This could negatively impact several biological pathways simultaneously, potentially leading to severe consequences for the cell’s survival.

  • Potential for Unintended Cross-Regulation

    • For instance, if similar binding sequences exist elsewhere in the genome, the regulator might mistakenly activate or repress genes not intended to be part of the regulon, leading to uncoordinated gene expression.

T/F: Transcription regulation in Archaea more closely resembles that of Eukarya than Bacteria.

FALSE

The regulation of transcription in Archaea more closely resembles that of Bacteria.

Inducible or repressible?

NrpR operon

Inducible

The (+) and (-) regulation systems in Archaea controls the binding of

• RNA polymerase

• TBP or TFB

Explain NrpR protein as archaeal repressor in nitrogen metabolism

• NrpR protein acts as archaeal repressor in nitrogen metabolism because in the presence of ammonia, it will bind to BRE, TATA in promoter, physically blocking access of RNA polymerase to DNA and preventing transcription of genes for nitrogen metabolism (since there’s no need to metabolize nitrogen in the presence of ammonia).

• Meanwhile, in the shortage of ammonia, a-Ketoglutarate will not be converted into glutamate and, upon its accumulation, will bind to NrpR protein, causing the NrpR protein from being released in DNA and subsequently allowing binding of TBP, TBF to promoter, recruiting RNA polymerase and beginning transcription.

  • (Makes sense since in shortage of ammonia, there is a need to metabolize nitrogen and convert into ammonia)

NrpR protein functions as repressor in nitrogen metabolism of which archaeal species?

Methanococcus maripaludis

Explain how TrmBL1 can act as both repressor and activator in archaea

• TrmBL1 as repressor in sugar uptake operon

  • Without maltose, TrmBL1 remains bound to region near promoter (BRE, TATA), preventing binding of TBP, TFB to BRE, TATA and recruitment of RNA polymerase.

    • No transcription of sugar uptake genes (since there is no sugar to uptake anyway)

  • In the presence of maltose, TrmBL1 binds to maltose, releasing itself from DNA and thus allowing binding of TBP, TFB to BRE, TATA and recruiting RNA polymerase, subsequently beginning transcription of sugar uptake genes.

    • There is transcriptipn of sugar uptake genes since there is sugar to uptake.

• TrmBL1 as activator in glucose synthesis operon

  • Without maltose, TrmBL1 binds upstream of BRE, TATA, allowing binding of TFB, TBP and aiding recruitment of RNA polymerase, beginning transcription of genes for glucose synthesis.

    • No glucose so glucose has to be synthesized!

  • With maltose, TrmBL1, instead of binding upstream and aiding RNA polymerase recruitment, binds to maltose.

    • Release of TrmBL1 from DNA causes dissociation of components of initiation complex, including TBP, TFB, RNA polymerase.

    • No need to synthesize glucose since there is glucose!

T/F: Some archaeal regulators function as both repressor and activator

TRUE

_ are universally encoded transcription elongation factors in archaeal genomes

SPT4/SPT5 complexes

T/F: Some archaeal proteins interact with DNA not in promoter or activator r

TRUE

Archaeal Spt4/5 secures RNA polymerase onto the DNA template when the initiation complex transitions to the elongation complex, rendering the elongation complex more stable.

Explain how SPT4/5 functions

• SPT4/5 complex does not bind at typical regulatory spots (promoters, activator regions) but instead binds to RNA polymerase during elongation phase of transcription.

• Helps in stabilizing RNA polymerase by ensuring it stays attached to DNA and continues transcription efficiently.

_ regulon system of E. coli consists of 10 genes located in five operons that encode proteins involved in the uptake of maltose and short maltodextrins.

Maltose/maltodextrin regulon system

T/F Enzyme repression typically refers to catabolic enzymes that are turned off by the final product of a biosynthetic pathway.

False
Justification: Enzyme repression usually involves anabolic enzymes where the final product acts as a corepressor, turning off the enzyme.

T/F Positive control mechanisms always require the presence of inducers to enhance the activity of transcription factors.

True
Justification: Positive control mechanisms depend on inducers to activate transcription factors, thereby promoting gene expression.