bio midterm2

Lambda phage lifestyle decision

Lambda phage can enter either a lytic or lysogenic cycle depending on environmental conditions and molecular signals. The decision is made through the competition between Cro protein and λ repressor protein for binding to operator sequences, which ultimately determines whether the phage will replicate and lyse the host or integrate into the host genome.

Function of antiterminator protein N

The N protein is an antiterminator that binds to transcription-terminating sequences in the lambda genome and allows RNA polymerase to transcribe delayed early genes. This action is essential for enabling either lysogeny or lytic pathways to proceed, depending on subsequent molecular events.

Role of Cro protein

Cro protein is a repressor that promotes the lytic cycle by binding to operator sequences and preventing transcription of the cI gene, thereby inhibiting λ repressor production. Cro protein enhances its own transcription and the expression of genes needed for host cell lysis and phage assembly.

Role of λ repressor protein (CI)

The λ repressor protein, encoded by the cI gene, promotes lysogeny by binding cooperatively to operator sequences, activating its own transcription from the pRM promoter, and repressing cro transcription. This maintains a stable lysogenic state with minimal phage gene expression.

Function of cII and cIII proteins

cII and cIII proteins form a complex that activates the pRE promoter, initiating transcription of the cI gene to produce λ repressor. The stability of cII is critical and depends on environmental conditions—protease activity degrades cII under favorable conditions, favoring the lytic cycle.

Impact of bacterial growth conditions

When bacteria are actively growing, high protease activity degrades cII, reducing λ repressor levels and favoring the lytic cycle. Under poor growth conditions, low protease activity allows cII to persist, promoting lysogeny by enabling repressor production.

Switch from lysogeny to lytic cycle (induction)

Induction ends lysogeny and restarts the lytic cycle, often triggered by DNA damage such as UV light. Activated RecA cleaves λ repressor proteins, removing them from operator DNA, allowing Cro expression and prophage excision.

Structure and function of λ repressor

The λ repressor is a homodimer formed from two 236-amino acid monomers, with domains that bind to 17-bp operator DNA. It blocks cro transcription while enhancing its own expression, maintaining the lysogenic state until inactivated.

Role of Q protein in lytic cycle

Q protein is a positive regulator that promotes transcription of late genes in the lytic cycle, including those required for phage assembly and host cell lysis. Its expression is facilitated when Cro protein prevails over λ repressor protein.

What is a riboswitch?

A riboswitch is a segment of an mRNA molecule that binds a small regulatory molecule, resulting in changes to gene expression.

Where are riboswitches most commonly found?

They are common in bacteria, regulating about 5% of bacterial genes, and also found in archaea, fungi, algae, and plants.

What processes can riboswitches regulate?

Transcription, translation, and mRNA stability.

How do riboswitches regulate transcription in B. subtilis?

Low TPP levels allow formation of an antitermination stem loop, enabling transcription of thi operon genes; high TPP levels bind the riboswitch and cause formation of a termination stem loop, halting transcription.

What is the role of TPP in riboswitch-mediated regulation?

TPP (thiamin pyrophosphate) binds to riboswitches to regulate the expression of genes involved in its own biosynthesis.

What is the outcome of high TPP concentration in B. subtilis?

TPP binds to the riboswitch, forming a termination stem loop followed by a poly-U tail, causing intrinsic termination of transcription.

What is the outcome of low TPP concentration in B. subtilis?

Antitermination stem loops form, allowing RNA polymerase to transcribe the operon and produce mRNA for TPP synthesis enzymes.

How does riboswitch regulation differ in E. coli for TPP synthesis?

In E. coli, the thiMD operon is regulated at the level of translation, not transcription.

How is the Shine–Dalgarno sequence involved in riboswitch translation regulation?

At low TPP, an antisequestor stem loop exposes the Shine–Dalgarno sequence for ribosome binding; at high TPP, a stem loop forms that hides it, preventing translation.

What happens to thiMD mRNA at high TPP concentration in E. coli?

TPP binds the riboswitch, inducing formation of a stem loop that hides the Shine–Dalgarno sequence and start codon, blocking translation.

What does the thiMD operon do?

It contains genes for enzymes involved in TPP biosynthesis in E. coli.

What riboswitch mechanism regulates mRNA stability?

Riboswitches can bind small molecules to trigger mRNA cleavage, thereby preventing translation.

How is the glmS gene regulated in B. subtilis?

At low GlcN6P, the glmS gene is transcribed and translated; at high GlcN6P, the metabolite binds the riboswitch and induces cleavage of the mRNA, preventing translation.

What does the glmS gene encode?

It encodes the enzyme glutamine:fructose-6-phosphate amidotransferase, involved in GlcN6P synthesis.

What happens when GlcN6P binds the glmS riboswitch?

It triggers self-cleavage of the mRNA, destabilizing it and preventing translation.

How do riboswitches contribute to cellular efficiency?

They help cells conserve energy and resources by quickly adjusting gene expression based on metabolite availability.

What is a common structural feature of riboswitch-regulated mRNAs?

The 5' UTR contains sequences that can form stem loops in response to metabolite binding.

What is the function of the poly-U tail following some riboswitch stem loops?

It enables intrinsic transcription termination when paired with a preceding stem loop.

What type of control mechanism is the TPP riboswitch in B. subtilis an example of?

An attenuation mechanism that senses metabolite concentration to regulate transcription.

What is the significance of riboswitches in gene regulation?

They allow direct sensing of metabolite levels without the need for protein intermediates, enabling rapid and specific regulation of gene expression.

What are the two main modes of gene regulation in bacteria?

Transcriptional regulation (most common) and translational regulation.

What is the role of sigma factors in bacterial transcription?

Sigma factors guide RNA polymerase to specific promoter sequences, controlling which genes are transcribed.

How does E. coli respond to heat stress at the transcriptional level?

By expressing an alternative sigma factor (σ^32, encoded by rpoH), which allows transcription of heat shock and chaperone genes.

What is the normal sigma factor used under typical conditions in E. coli?

σ^70, which guides RNA polymerase to -10 Pribnow box-rich promoters.

What is σ^32 and what is its role?

It is an alternative sigma factor activated during heat stress that directs RNA polymerase to transcribe heat shock genes.

How is σ^32 regulated under normal and heat shock conditions?

Under normal conditions, it is bound and inhibited by chaperone proteins; during heat shock, chaperones are redirected to damaged proteins, freeing σ^32 to activate heat response genes.

What is the rpoH gene?

It encodes the σ^32 sigma factor, which is involved in the heat shock response.

What changes occur in sigma factors during high heat?

σ^70 becomes unstable, while σ^32 and σ^E are upregulated and direct transcription of stress-response genes.

What do chaperone proteins do in heat shock conditions?

They are released from σ^32 and redirected to bind and refold or degrade heat-damaged proteins.

What is an example of sigma factor switching in response to environmental stress other than heat?

In Bacillus subtilis, poor conditions trigger sporulation by activating sporulation-specific sigma factors.

How does switching sigma factors impact gene expression?

It globally changes the pattern of transcription, silencing some genes and activating others specific to the stress.

What are the three main types of transcription regulation mechanisms in bacteria?

Operon-specific control, CAP–cAMP control, and alternative sigma factors.

What are the two mechanisms of translational regulation in bacteria?

Binding of translation repressor proteins and binding of antisense RNA to mRNA.

How do translation repressor proteins inhibit gene expression?

They bind near the Shine–Dalgarno sequence of mRNA, blocking ribosome binding and translation initiation.

How is ribosomal protein production regulated in E. coli?

One protein from each ribosomal operon can bind to the mRNA it was translated from and inhibit translation by blocking ribosome access.

What is antisense RNA?

An RNA molecule complementary to a portion of an mRNA that binds and prevents its translation by blocking ribosome access.

What is IS10 and what does it encode?

IS10 is an insertion sequence in bacteria that encodes the transposase enzyme required for transposition.

How is transposase expression regulated in IS10?

Through antisense RNA transcribed from a stronger promoter, which binds the transposase mRNA and blocks its translation.

Why is limiting transposase expression important?

Too much transposition can cause harmful mutations in essential genes, so expression must be tightly regulated.

Which promoter in IS10 is stronger, and what does it produce?

P_OUT is stronger and produces antisense RNA that blocks transposase translation.

What does antisense RNA do in the context of IS10 regulation?

It binds to the 5' end of transposase mRNA, covering the Shine–Dalgarno sequence and preventing translation.

How does bacteria ensure occasional transposase activity?

Some transposase mRNAs escape antisense binding, allowing low-level expression and rare transposition events.

Why is translational regulation useful for bacteria?

It allows quick and fine-tuned control over protein synthesis without needing to transcribe new mRNA.

What is a repressible operon?

An operon whose transcription can be inhibited by the presence of the end product it synthesizes, using a negative feedback mechanism.

How does attenuation differ from inducibility in gene regulation?

Inducibility acts like an on/off switch controlled by repressor binding, while attenuation functions like a dimmer, fine-tuning transcription based on metabolite availability.

What does the trp operon synthesize?

It encodes five structural genes (trpE, trpD, trpC, trpB, trpA) needed for tryptophan biosynthesis.

What is the function of trpR?

It encodes the Trp repressor protein, which requires tryptophan to become active and repress the operon.

How does tryptophan act in feedback inhibition?

It functions as a corepressor that binds to the Trp repressor protein, enabling it to bind the operator and block transcription.

What happens in the trp operon when tryptophan is absent?

The Trp repressor remains inactive and cannot bind the operator, allowing transcription of operon genes to occur.

What happens when tryptophan is present?

Tryptophan binds to and activates the repressor, which binds to the operator and prevents transcription of the operon genes.

Why is attenuation needed in addition to feedback inhibition in the trp operon?

Attenuation allows fine-tuning of transcription, adjusting tryptophan production to current cellular needs, even if repression is already in place.

What is the trpL region?

A leader sequence upstream of the structural genes containing four regions capable of forming stem-loops, including a 14-amino acid coding sequence.

What is the purpose of the two adjacent tryptophan codons in trpL?

They act as sensors of tryptophan availability, influencing whether transcription continues or terminates.

What stem-loop causes transcription termination in the trp operon?

The 3–4 stem loop, which is followed by a poly-U string and triggers intrinsic termination.

What stem-loop allows transcription to continue?

The 2–3 stem loop, which prevents formation of the 3–4 termination structure and allows transcription into the structural genes.

How does tryptophan abundance affect stem-loop formation?

When tryptophan is abundant, the ribosome quickly translates the leader peptide, allowing 3–4 loop formation and halting transcription.

How does tryptophan scarcity affect stem-loop formation?

The ribosome stalls at the Trp codons, allowing region 2 to pair with region 3, forming the 2–3 antitermination loop and permitting full transcription.

What role does transcription–translation coupling play in attenuation?

The speed of ribosome movement during translation of trpL mRNA determines which stem-loop forms and whether transcription continues.

What happens if the Trp codons in the attenuator are mutated to stop codons?

Translation halts early, ribosome can't block region 2, the 2–3 loop forms, and transcription proceeds constitutively regardless of tryptophan.

What happens if the Trp codons are mutated to Leu codons?

The system responds to leucine instead of tryptophan, attenuating transcription based on leucine availability instead.

What is the structure of the 3–4 stem loop?

It is a transcription termination signal formed by regions 3 and 4 followed by a poly-U tail, inducing intrinsic termination.

What is the function of the 1–2 stem loop in trpL?

It induces a temporary pause in transcription if no ribosome is bound, allowing time for attenuation decision-making.

What experimental evidence supports attenuation?

Mutations in Trp codons or in regions 3 and 4 alter attenuation efficiency, confirming their roles in sensing amino acid levels and regulating transcription.

What is the effect of mutations that disrupt base pairing between regions 3 and 4?

They reduce formation of the termination stem loop, decreasing repression and allowing more transcription.

How do other amino acid operons use attenuation?

Like the trp operon, they contain leader peptides with repeated codons for their specific amino acid and use stem-loop formation to regulate transcription.

Name three other amino acid operons that use attenuation.

Histidine, phenylalanine, and leucine operons—all use codon-rich leader peptides and stem-loop regulatory mechanisms.

What does the term "antitermination" mean in this context?

It refers to a stem-loop structure (2–3) that prevents transcription termination and allows full operon gene transcription.

How does the 3–4 stem loop terminate transcription?

It causes RNA polymerase to pause and detach at a poly-U stretch, an intrinsic termination mechanism.

Why is attenuation considered fine-tuned regulation?

It adjusts operon gene transcription dynamically based on small changes in metabolite (e.g., tryptophan) levels.

What did Jacob and Monod's mutational analysis of the lac operon reveal?

It identified the functions of lac operon genes and regulatory regions and led to the discovery of the operon's transcriptional control mechanisms.

What is the lacI− mutation?

A mutation in the lacI gene that produces a repressor incapable of binding the operator, resulting in constitutive transcription.

What is the lacIs mutation (super-repressor)?

A mutation in the lacI gene where the repressor cannot bind allolactose, preventing its release from the operator and causing noninducible transcription.

What is the lacZ− mutation?

A mutation in the lacZ gene resulting in no production of β-galactosidase.

What is the lacY− mutation?

A mutation in the lacY gene that prevents production of permease.

What is a lacOc mutation?

A mutation in the operator (lacO) that prevents binding by the repressor protein, leading to constitutive transcription of operon genes.

What is a lacP− mutation?

A mutation in the promoter region that prevents RNA polymerase binding, resulting in no transcription of operon genes.

What type of mutations affect translation of the lac operon polycistronic mRNA?

Nonsense (polar) mutations in lacZ can stop ribosomes early, preventing translation of lacY and lacA due to lack of additional Shine–Dalgarno sequences.

What does cis-acting mean in the context of lacO mutations?

The mutation only affects genes on the same DNA molecule and cannot influence genes on another chromosome or plasmid.

What does trans-acting mean in the context of lacI?

The lacI gene product (repressor protein) can diffuse and regulate operons on other DNA molecules, such as plasmids or chromosomes.

How can complementation help identify mutated genes in the lac operon?

By introducing functional copies of specific genes into mutants and analyzing whether wild-type phenotypes are restored.

What does constitutive transcription mean?

Continuous, unregulated transcription regardless of inducer presence, due to mutations in the repressor or operator.

What does inducible transcription mean?

Transcription that occurs only when an inducer (allolactose) is present to inactivate the repressor protein.

What does noninducible mean in lac operon regulation?

The operon cannot be activated by lactose; often due to super-repressor (lacIs) or promoter (lacP−) mutations.

What is the function of the Shine–Dalgarno sequence in the lac operon?

It's the ribosome-binding site upstream of lacZ; if translation stops early, downstream genes like lacY won't be translated due to lack of separate binding sites.

What is the effect of a lacZ− / lacY+ partial diploid?

Complementation occurs; β-galactosidase is provided by the plasmid copy and permease by the chromosome, restoring lactose metabolism.

How does lacOc affect lacZ+ in cis?

It causes constitutive transcription of lacZ+ only when they are on the same DNA molecule, demonstrating its cis-acting nature.

How does lacI+ restore inducible control in partial diploids with lacI−?

Because lacI+ produces functional repressor protein that is trans-acting and can regulate operons on different DNA molecules.

Why is the lac repressor a homotetramer?

It consists of four identical subunits that enable binding to multiple operator regions and allow DNA looping to block transcription.

What is DNA looping in lac operon regulation?

A structure formed when repressor protein binds to operator sites O1 and O3, pulling the DNA into a loop and blocking RNA polymerase access.

What happens when operator region O1 is mutated (lacOc)?

The twofold symmetry is disrupted, repressor binding is blocked, DNA looping can't form, and transcription becomes constitutive.

What is the primary operator region required for repressor binding?

O1; binding here is necessary for repressor interaction with O2 or O3 and for DNA looping to repress transcription.