Molecular Bio 330 Exam 3 - Lec 4-6

In the Trp operon, what occurs when tryptophan levels are abundant?

The Trp repressor binds to the operator and represses the operon, so no Trp structural gene expression should occur.

In the Trp operon, what occurs when tryptophan levels are absent?

The Trp repressor does not bind to the operator and therefore does not repress the Trp operon and it will be expressed

What specific site within the Trp operon leader sequence is responsible for premature transcription termination?

The attenuator.

What causes transcription to be initiated even when trp repressor is active?

The leaky expression of Trp structural genes

How does the $Trp$ repressor's magnitude of transcriptional reduction compare to that of attenuation?

The repressor reduces transcription 70 fold, while attenuation provides an additional 8~10 fold reduction.

Under what condition does a ribosome stall at the tryptophan codons in the leader sequence?

When tryptophan levels are low and charged tRNA is unavailable.

Which secondary structure forms in the leader mRNA when tryptophan levels are low, allowing transcription to proceed?

The 2-3 stem-loop (antiterminator hairpin).

Which secondary structure forms in the leader mRNA when tryptophan levels are high, causing transcription to terminate?

The 3-4 stem-loop (terminator hairpin).

How does attenuation use translation to control transcription in prokaryotes?

The position of the ribosome on the leader sequence determines which RNA secondary structure forms.

Besides the Trp operon, name one other amino acid operon that utilizes an attenuation mechanism.

The Histidine operon (also Threonine, Leucine, or Phenylalanine).

What is the fundamental reason that different cell types in a multicellular organism have different functions?

They express different sets of proteins despite containing the same DNA

Eukaryotic gene expression is influenced by?

Chromatin modifications

Eukaryotic transcription is regulated by?

Trans-acting transcription factors that bind to cis-acting sites

In Eukaryotes, DNA must unwind from the histone proteins before?

Transcription

Are eukaryotes monocistronic or polycistronic?

Monocistronic because each structural gene has its own promoter and is transcribed separately

What is the term for the complex of DNA, histones, and nonhistone proteins found in the eukaryotic nucleus?

Chromatin.

What is the basic structural unit of chromatin?

Nucleosome

A histone octamer consists of two copies each of which four core histone proteins?

H2A, H2B, H3, and H4.

What is a histone?

Eight-protein complex found at the center of a nucleosome core particle that prevents DNA from becoming tangled and protects it from DNA damage

Why do histones form strong electrostatic bonds with the phosphate groups of DNA

Histones are positively charged (basic) proteins, while DNA is negatively charged.

Which histone protein is responsible for stabilizing higher-order chromatin architecture (pulls nucleosomes together into a regular repeating array) but is not part of the octameric core?

Histone H1

To allow gene expression in eukaryotes, chromatin must:

• Relax compact structure

• Expose regions of DNA to regulatory proteins

• Have a reversal mechanism for inactivity

How does chromatin remodel?

Chromatin-remodeling complex which binds directly to DNA sites and reposition nucleosomes which can make chromosome regions accessible to other proteins in the cell

Where do histone tails protrude relative to the nucleosome core?

They pass through the minor groove of the DNA and extend outward.

Which enzyme is responsible for adding acetyl groups to the lysine residues of histone tails?

Histone acetyltransferase (HAT).

How does histone acetylation affect the chemical charge of the histone protein?

It neutralizes the positive charge of the amino group on the lysine side chain

What creates euchromatin?

When histone is neutralized upon acetylation

What is euchromatin?

Loosely packed, transcriptionally active form of chromatin that is intact with histone acetylation of lysine residues

In the model organism $Arabidopsis\ thaliana$, what is the function of the Flowering locus $C$ ($FLC$) gene?

It encodes a transcription factor that represses flowering.

How does the Flowering locus D (FLD) gene promote flowering in Arabidopsis?

It encodes a protein (histone deacetylase activity) that promotes FLC repression

How does the Flowering locus C (FLC) gene repress flowering in Arabidopsis?

It encodes a transcription factor/regulator (repressor) that represses flowering

High FLC vs Low FLC

High FLC: late flowering

Low FLC: early flowering

Active FLD vs Inactive FLD

Active FLD: Reduced FLC → Earlier flowering

Inactive FLD: High FLC → Late flowering

In eukaryotic gene regulation, what are 'cis-acting sites'?

DNA sequences (like promoters and enhancers) that serve as control elements for nearby genes.

What are 'trans-acting factors' in the context of transcription?

Regulatory proteins, such as transcription factors, that bind to DNA control elements.

Which general transcription factor consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs)?

TFIID

What is the TATA-binding protein (TBP)?

Determines transcription start site

What is TBP-associated factors (TAF)?

Help TBP recognize different DNA promoters and stabilize the TFIID complex + many more

What are transcription factors (TF)?

Proteins that help turn specific genes “on” or “off” by binding to nearby DNA

Which transcription factor remains bound through multiple rounds of transcription initiation?

TFIID

As long as TFIID remains bound to the promoter, what happens?

Additional RNA polymerases may be able to attach for additional rounds of transcription

What are regulatory (control) elements?

Segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription of neighboring genes

What is a promoter?

DNA sequence typically located upstream a gene that serves as the binding site for RNA polymerase and transcription factors

Which eukaryotic general transcription factor possesses both protein kinase and DNA helicase activities and is the only GTF with enzymatic activity?

TFIIH

What is the function of the helicase subunit of TFIIH?

It pries apart the DNA double helix to create the transcription bubble via ATP hydrolysis which exposes the template strand of the gene

The preinitiation complex of GTFs and RNA polymerase II assembles at the?

TATA Box

What are examples of GTFs?

TFIID and TFIIH

What distinguishes eukaryotic mRNA from prokaryotic mRNA regarding the number of genes encoded per transcript?

Eukaryotic mRNA is typically monocistronic, while prokaryotic mRNA is often polycistronic.

Which type of histone modification is specifically associated with the formation of euchromatin?

Acetylation.

In eukaryotic cells, transcription occurs in the nucleus while translation occurs in the _____.

Cytoplasm (or Cytosol).

Which enzyme adds methyl groups to arginine and lysine residues in histones?

Methyltransferase.

HDAC inhibitors.

Chemical compounds that prevent deacetylation, leading to increased histone acetylation and altered gene expression.

What effect does the phosphorylation of serine residues on RNA pol II have on the transcription process?

It triggers movement downstream and attracts mRNA processing proteins.

In the Trp operon attenuation model, if the ribosome moves into the 1-2 region, what is prevented from forming?

The 2-3 stem-loop.

Eukaryotic transcription factors that increase the rate of transcription are called _____.

Activators.

How does histone deacetylation affect the 'open' or 'closed' configuration of chromatin?

It promotes a 'closed' configuration, inhibiting transcription.

What are the posttranscriptional modifications of RNA processing (eukaryotes)?

• Addition of 5’ cap (7-mG cap)

• Addition of 3’ tail (poly-A tail)

• Excision of introns (splicing)

Which RNA polymerase is responsible for the transcription of all protein-coding genes in eukaryotes?

RNA Polymerase II.

What is the C-terminal domain?

Serves as a scaffold for the organization of factors involved in processing pre-mRNAs

C-terminal domain processing includes?

• Capping

• Polyadenylation

• Intron removal

What do activators do?

• Bind to special DNA regions called enhancers

• Help increase the rate of transcription

What are enhancers?

DNA regions containing distal control elements that increase transcription when activators bind.

Even if the enhancer is far away from the gene, how can it work?

The DNA bends or loops so that the activator at the enhancer can physically interact with the promoter

What type of genes require specific transcription factors for high levels of transcription?

Genes that are not expressed all the time

What type of protein commonly acts as a specific transcription factor that increases transcription?

Activator proteins

Regulatory factors are?

Proteins (like transcription factors) that control gene expression

What do regulatory factors bind to?

Bind to regulatory DNA sequences such as promoters, enhancers, or operators

Regulatory factor binding helps determine?

Whether a gene is turned on or off

Regulatory (transcription) factors vs Regulatory (control) elements?

• Regulatory (transcription) factors are proteins that control transcription by binding to DNA

• Regulatory (control) elements are specific regions of DNA where the proteins bind

What is the primary function of the lac operator in prokaryotic gene regulation?

It serves as the binding site for the lac repressor to block transcription.

Under what specific condition does the lac repressor undergo a conformational change to release its grip on the operator?

When lactose (allolactose) binds to the repressor.

For the lac operon to be optimally expressed, what are the necessary statuses of glucose and lactose?

Glucose must be absent and lactose must be present.

What is the transcriptional state of the lac operon when both glucose and lactose are present?

Operon OFF (or expressed at very low basal levels).

In an I^- mutant of the lac operon, why are the structural genes always turned on?

The repressor protein is altered or absent and cannot bind to the operator, so the structural gene will NOT be repressed

What is the effect of an O^c (constitutive operator) mutation on lac operon regulation?

The operator DNA sequence is altered so the normal repressor cannot bind, leading to constitutive expression, and the genes are always on

How does an I^s (superrepressor) mutation affect the lac operon?

The repressor cannot be inactivated by an inducer, keeping the operon permanently repressed and always off

How many structural genes are contained within the trp operon of E. coli?

Five (trpE, trpD, trpC, trpB, trpA).

What molecule acts as the corepressor for the trp operon?

Tryptophan.

If tryptophan is available in the environment, E. coli will?

Take it up and use it to build proteins

If tryptophan is not available, in the environment, E. coli can?

Make their own tryptophan by producing enzymes required for the biosynthesis of tryptophan

Under what condition does the trp repressor become active and bind to the operator?

Only when tryptophan is bound to the repressor protein.

When tryptophan levels are low in the cell, what is the status of the trp repressor?

The repressor is inactive and cannot bind to the operator.

Define 'attenuation' in the context of the trp operon.

A regulatory mechanism that reduces transcription when tryptophan levels are high by prematurely terminating the mRNA.

Why is attenuation unique to prokaryotes (bacteria)?

It requires transcription and translation to occur simultaneously in the same compartment.

What is trpP vs trpP?

trpP: promoter — binding site for RNA polymerase

trpO: operator — binds repressor (binding site for a repressor protein)

What do leader sequences do?

Encode a short polypeptide and also contains an attenuator

What has its own promoter and other regulatory sequences, is not part of the trp operaon, constitutively expressed, and is encoded by trpR?

trp repressor protein

Why is tryptophan considered a corepressor?

It switches an inactive repressor into its active site

What acts as an inducer by binding to the lac repressor, inactivating it, and allowing transcription of the lac operon?

Lactose/Allolactose

Intrinsic termination (Rho-independent) occurs?

When a termination sequence is transcribed into RNA, causing the newly found transcript to fold back on itself, forming a hairpin structure

What happens during intrinsic termination (rho-independent)?

• mRNA forms a stable secondary structure hairpin loop (stem-loop) → many G and C

• RNA hairpin is followed by multiple uracil nucleotides

• The bonds between uracil and adenine are very weak lower the energy of destabilization for the RNA-DNA duplex

• RNA structure and sequences (hair pin/stem-loop and uracil sequences) → terminates transcription

What are the three binding sites for tRNA on a ribosome? What do they do?

• A site: holds (aminoacyl tRNA binding site; accept) the tRNA that carries the next amino acid to be added to the chain

• P site: holds the tRNA with the growing polypeptide chain (peptidyl-tRNA binding site) + holds the first initiator tRNA

• E site: the exit site, where discharged tRNAs leave the ribosome

The trp operon repressor has two binding sites. In sequential order, to what two things does the repressor bind?

Tryptophan and operator

Which two regions of the trp leader mRNA pair to form the 'terminator' hairpin?

Region 3 and Region 4.

Which two regions of the trp leader mRNA pair to form the 'antiterminator' hairpin?

Region 2 and Region 3.

What feature is located within Region 1 of the trp leader sequence that 'senses' tryptophan levels?

Two adjacent Trp codons.

In the trp operon, what happens to the ribosome when tryptophan levels are low?

The ribosome stalls at the Trp codons in Region 1.

What hairpin structure forms when the ribosome stalls at Region 1 of the trp leader mRNA?

The 2-3 antiterminator hairpin.

If the ribosome covers Region 2 of the trp leader mRNA, which hairpin is forced to form?

The 3-4 terminator hairpin.

Formation of the 3-4 hairpin in the trp operon leads to the termination of _________

transcription