Gene Expression Regulation, Epigenetics, and Protein Processing in Molecular Biology

How do enhancers affect gene expression?

Bind tissue-specific TFs.

Recruit histone acetyltransferases

Stabilize transcription initiation.

CAV1 gene is not transcribed:

* Two molecular reasons that could prevent CAV1 from being transcribed?

*"All other genes in the osteoblast are appropriately transcribed." What potential reasons are ruled out by this statement?

Two reasons:

1. Mutated/methylated enhancer or promoter.

2. Condensed chromatin.

Ruled out: RNA Pol II or general TF issues.

To investigate... not turn green (GFP is not expressed)..

What does this result indicate?

Does it matter if the CAV1 enhancer-GFP reporter gene is tested in a normal or diseased osteoblast?

If the osteoblast with your reporter gene had turned green, what could you have concluded?

No GFP = Enhancer defective.

Cell type doesn't matter.

If GFP = promoter/TF/chromatin issue.

CAV1 transcribed but NOT translated.

What are three molecular reasons why this would happen?

Select one of the reasons you listed and describe why translation would not occur in this scenario?

Three reasons:

1. mRNA lacks 5' cap -> can't recruit initiation factors.

2. Short or missing polyA tail -> unstable transcript.

3. miRNA binding to 3'UTR or presence of premature stop codon triggering NMD.

Example: Without a proper 5' cap, eIFs and the ribosome cannot assemble at the 5'UTR.

NCAM is a gene that is normally only transcribed in neurons. Are neurons the only cell type with the NCAM gene? If not, what is the molecular mechanism that causes NCAM to only be expressed in neurons.

All cells have NCAM gene.

Neurons express neuron-specific TFs.

What are the ways that you could induce (force) expression of NCAM in muscle cells?

Add neuron TFs or mutate enhancer to bind existing TFs.

Why are CpG islands important for epigenetic memory?

Maintain methylation patterns = epigenetic memory.

If all the Guanines in a CpG island were mutated to Adenines or Uracils, how would that affect the ability of DNA methyltransferases to methylate this region?

DNA methyltransferases recognizes CpG dinucleotides; mutation disrupts this recognition, preventing methylation and destroying the ability to maintain epigenetic memory.

How does DNA methylation inhibit transcription?

Blocks TF binding

Recruits HDACs (Histone Deacetylases)

Condenses chromatin

How are the functions of Dnmt1 and Dnmt3 similar and how are they different?

Both methylate CpG

Dnmt1 = maintenance

Dnmt3 = new marks.

A protein is produced in two different cells. The primary structure is the same in both cells, but the amount of peptide is greater in one cell. What could be happening to the mRNA transcript in the cell in which the amount of peptide is greater?

Longer polyA tail = stable mRNA = more translation occurring.

A gene called TBK2 has different splicing isoforms found in different cell types, including intestinal epithelial cells, white blood cells, and skeletal muscle cells. What mechanism allows different splicing isoforms to be expressed in the different cell types?

Alternative splicing by cell=specific splicing factors.

In another cell, TBK2 is transcribed, but the mRNA for TBK2 is never exported from the nucleus. What are the possible explanations for this?

Missing cap/tail/EJCs -> incomplete processing

What happened to unexported mRNA in the nucleus?

Degraded by nuclear exonucleases.

What are the four functions of the 5' cap?

1. Splicing

2. Export

3. Translation

4. Protection

Does the 5' cap influence splicing? If so How?

Yes, the CBC (Cap binding complex) helps splice first intron

The same gene is transcribed in two different cells, but the mRNA sequences produced from this gene are different in each cell. What are possible explanations?

Alternative splicing, alternative polyA site, or RNA editing.

Defensin is a defense-related gene that is translated into a protein (also called Defensin) in macrophages when they are exposed to pathogens. Describe how defensin expression is normally suppressed in the absence of a bacterial infection. Your answer should include whether defensin transcription or translation is affected, the name of the processing event that is influenced by infection & what specifically changes during infection to increase the production of the Defensin protein.

Without infection: mRNA unspliced -> no translation.

During infection: splicing activated -> mature mRNA -> translated to Defensin.

Regulation occurs at RNA splicing.

Building on your answer from #18, you are treating a patient with an immunodeficiency who has a severe bacterial infection. You notice that although their macrophages are transcribing defensin mRNA, the mRNA is not being translated. How is this problematic for this patient? Help this individual by diagnosing what could be going wrong at the molecular level.

Translation blocked (miRNA inhibition or defective initiation factor). Without defensin protein, improper defense against bacteria.

Two peptides have almost the exact same primary structure, except that one has 10 fewer amino acids at the carboxy-terminus (C-terminus) of the protein. What is a possible explanation for the protein with 10 fewer amino acids?

Alternative polyA site or early stop codon.

21. You are analyzing two different cells that transcribe the same gene called neurexin (NRXN). The initial NRXN primary mRNA transcript (prior to processing) is identical in the two cells, but the mRNA that exits the nucleus to be translated in the cytosol is composed of different combinations of exons. The two different mRNA transcripts now encode proteins with differences in their amino acid sequences.

* How were different versions of the NRXN protein produced in the cells? (Your answer should include the names of the proteins and sequences involved.)

* What are the two different versions of the NRXN protein are referred to as?

Alternative splicing by cell-specific splicing factors binding splicing enhancers/silencers.

The versions are called isoforms.

A gene was successfully transcribed into mRNA, and then exported into the cytosol. But the polypeptide it encodes is not successfully translated. Describe the events required for successful translation in the cytosol.

Cap binding complex (CBC) + eIFs (eukaryotic initiation factors) binds 5' -> PABP (PolyA binding protein) at 3' -> circularize mRNA -> Ribosome binds, scans for AUG -> elongation begins.

If the mRNA described above was not successfully translated because it did not circularize, which proteins could have misfunctioned?

eIF4E, eIF4G, or PABP malfunction.

An mRNA is exported from the nucleus, but not circularized. What could prevent circularization? What would happen to this mRNA?

Missing cap/tail -> unstable -> degraded.

The mRNA transcript encoded by a gene called TLR4 has 3 EJCs (Exon Junction Complexes) bound to it, while the mRNA transcript encoded by a gene called RBBP5 has 8 EJCs bound to it. What does this tell you about these two mRNAs?

RBBP5 underwent more splicing -> contains more exons/introns.

Describe the details of Nonsense Mediated Decay.

Stop codon before EJC -> UPF proteins degrade mRNA

Describe the molecular process known to increase the stability of casein mRNA in nursing mothers. How does this affect the levels of casein peptide produced?

Prolactin -> longer polyA tail -> more stable -> more protein.

You are an OBGYN doctor meeting with a patient who just gave birth. You find that the nursing mother is producing very low levels of casein in her breast milk, instead of the high levels of casein that are expected in nursing mothers. Based on what you learned about this process in BIO121, what is a likely explanation?

Prolactin or tail extension defect -> unstable mRNA -> low protein

Describe the critical importance of chaperone proteins. Do you think a cell could survive without chaperone proteins? Why/why not?

Helps folding and prevents aggregation. (Hsp70, Hsp60, calnexin). Without them, misfolded proteins accumulate and can kill the cell.

A peptide is being translated in the cytosol and immediately begins to misfold (during translation) because its hydrophobic residues start to aggregate with other hydrophobic residues. In this case, what protein is not functioning properly?

Hsp70 malfunction causes hydrophobic clumping.

After attempting to fold, the protein described in the previous question was degraded by a ___________________ because too many __________________ residues were located on the exterior of the protein (facing the cytosol) after folding.

Proteasome; exposed hydrophobic

Why is it so critical for misfolded proteins to be degraded?

Prevent toxic aggregates and avoid disease.

Insulin is an example of a protein that is proteolytically cleaved. What are the outcomes of Insulin proteolysis? Is this required for its activity? Is it reversible? Is proteolysis ever reversible?

Proinsulin is cleaved into insulin + C-peptide

Required for activity, irreversible, and proteolysis is never reversible.

What are microRNAs (miRNAs) known for? What parts of a pre-mRNA transcript do miRNAs usually bind to and what are the potential consequences? (i.e., what are the different ways that miRNAs do their job?)

They Bind 3'UTR and block translation. Recruit exonucleases and proteases to destroy bad proteins.

What would happen within a cell if it had a premature termination codon in the gene encoding the ER protein translocator? (What would happen to the ER protein translocator and how might that affect other proteins in the cell?)

Triggers NMD (Nonsense-mediated decay) -> No translocator -> ER proteins mislocalized.

You are studying a disease that occurs in individuals when an ER transmembrane protein called D2R is not inserted into the ER membrane of neurons. You find that the D2R gene is transcribed and the mRNA is exported out of the nucleus. You also notice that all other ER transmembrane proteins are inserted into the ER membrane properly. Based on what you have learned about protein processing, what could be a cause of this disease?

Mutated start/stop transfer or signal recognition site protein doesn't enter ER -> remains in cytosol.

Actin filaments and collagen fibers are examples of polymers. A protein polymer is defined as a _______________________.

Repeated subunits.

When a protein is subjected to proteolysis, what are the potential outcomes for the protein?

Activation (insulin), inactivation, or degradation; irreversible.