Cell S&F Lecture 33

What is the "Wobble Position" in translation?

The 3rd nucleotide of an mRNA codon. It has flexible base-pairing rules, allowing one tRNA to recognize multiple codons.

Which base in a tRNA anticodon is the most flexible and can pair with U, C, or A?

Inosine (I)

(T or F) For translation, multiple tRNAs carrying different amino acids can recognize a single codon.

False

(T or F) For translation, a single tRNA can carry multiple amino acids.

False; each tRNA carries a specific amino acid.

(T or F) For translation, multiple tRNAs can carry the same amino acid.

True; multiple tRNAs can recognize the same amino acid due to codon degeneracy.

(T or F) For the genetic code, multiple amino acids can be encoded by the same codon.

False; each codon specifies only one amino acid.

(T or F) For the genetic code, multiple codons can encode the same amino acid.

True; this is known as codon redundancy, where different codons specify the same amino acid.

Suppresser tRNA

A type of tRNA that can recognize and bind to a stop codon, allowing the incorporation of an amino acid instead of terminating translation.

Nonsense- Mediated Decay

A cellular mechanism that degrades mRNA transcripts containing premature stop codons, preventing the production of truncated and potentially harmful proteins.

Exon Junction Complex

A protein complex that assembles at exon-exon boundaries after splicing. It plays a crucial role in mRNA export, stability, and translation.

Nonstop decay

A surveillance mechanism that targets and degrades mRNAs lacking a stop codon, preventing the accumulation of non-functional proteins.

The ER Signal Sequence (SS)

The N-terminal region of a protein destined for secretion or the Plasma Membrane (PM).

• Composition: 6–12 mostly hydrophobic amino acids.

• Function: Acts as a biological "zip code" to ensure the protein is sent to the Endoplasmic Reticulum (ER).

The Signal Recognition Particle (SRP) steps

1. As the ribosome translates the mRNA, the SRP binds to the Signal Sequence as it emerges.

2. The SRP temporarily pauses translation to prevent the protein from folding in the cytosol.

3. The SRP docks the entire ribosome complex onto an SRP receptor on the ER membrane.

The Translocon and GTP Energy

• The Channel: The ribosome-SRP complex is handed off to the Translocon (a protein-conducting pore).

• Opening the Gate: GTP hydrolysis provides the energy required to transfer the Signal Sequence into the translocon and open the channel.

• Protein Entry: The elongating protein enters the ER co-translationally (meaning it's being made and imported at the same time).

Cleavage and Translocation of the Protein

1. The polypeptide enters the ER lumen in an unfolded state.

2. An enzyme called Signal Peptidase (or signal protease) recognizes the SS and cleaves it from the growing protein.

3. The SRP is recycled back to the cytosol after GTP is hydrolyzed to GDP.

Final Protein Release

1. Synthesis Ends: Once the stop codon is reached, the ribosome detaches from the translocon.

2. Result: The completed polypeptide is released entirely into the ER lumen.

3. Next Steps: The protein will now begin to fold into its functional 3D shape and move toward the Golgi apparatus for further sorting.

“Start Transfer” Sequence

A hydrophobic signal sequence that targets proteins to the membrane of the endoplasmic reticulum, initiating their insertion into the translocon during protein synthesis.

Where does a protein end up if it has an N-terminal signal peptide but NO other start or stop-transfer sequences?

It becomes a soluble protein in the ER lumen.

• Mechanism: The signal peptide starts translocation, but since there are no "stop" signals, the entire protein is pushed into the ER. The signal peptide is cleaved off, leaving the protein free-floating.

• Final destination: Secreted from the cell or staying inside an organelle.

What is the result of a protein having an N-terminal signal peptide AND an internal stop-transfer sequence?

It becomes a Single-pass Transmembrane Protein with the N-terminus in the ER lumen and the C-terminus in the cytosol.

• Mechanism: Translocation starts at the N-terminus. When the hydrophobic stop-transfer sequence hits the translocon, the channel discharges the protein sideways into the lipid bilayer.

• Key Detail: The N-terminal signal is cleaved.

What happens to a protein with a single internal start-transfer sequence (and no N-terminal signal)?

It becomes a Single-pass Transmembrane Protein where the internal sequence acts as the anchor.

• Mechanism: The internal sequence is recognized by SRP and brings the ribosome to the ER. The sequence itself stays embedded in the membrane as the transmembrane domain.

• Key Difference: The start-transfer sequence is NOT cleaved. One end of the protein stays in the cytosol while the other is pushed into the lumen.

How are multi-pass transmembrane proteins (like G-protein coupled receptors) woven into the ER membrane?

Through alternating Internal Start-Transfer and Stop-Transfer sequences.

• Start-Transfer: Initiates the "stitch" (insertion) into the translocon.

• Stop-Transfer: Halts the translocation and discharges the polypeptide into the membrane.

• Result: The protein "zig-zags" back and forth across the lipid bilayer.

In a multi-pass protein diagram, what determines the final orientation (location of N-terminal and C-terminal)?

The first signal sequence and the number of passes.

• If it starts with an N-terminal signal peptide (cleaved), the N-terminus ends up in the Lumen.

• If it starts with an Internal Start-Transfer (not cleaved), the N-terminus ends up in the Cytosol.

• Each subsequent Start/Stop pair adds two passes, effectively "stitching" the protein in place.

In eukaryotic gene expression, what three questions does regulation answer for a cell?

1. When? (Developmental timing)

2. Where? (Tissue differentiation)

3. How much? (Dosage compensation)

What are the two primary levels of gene regulation that occur inside the nucleus before mRNA is processed?

1. Genomic Level: Chromatin decondensation, DNA methylation, and histone acetylation (making DNA accessible).

2. Transcriptional Level: Control by transcription factors (deciding if mRNA is actually made).

What happens during RNA processing and nuclear export to regulate gene expression?

• RNA Splicing: Removing introns and joining exons.

• Export: Controlling the transport of mRNA from the nucleus to the cytoplasm.

How is gene expression regulated after mRNA reaches the cytoplasm?

1. Translation: Control by initiation factors, translational repressors, and microRNAs.

2. mRNA Degradation:Controlling how long the mRNA "lives" (turnover).

3. Post-translation: Protein folding, cleavage, chemical modification, and eventually protein degradation.

What is the purpose of DNA rearrangement (V(D)J recombination) in lymphocytes, and what are the gene segments involved in Heavy vs. Light chains?

• Goal: To produce billions of unique antibodies using a limited number of genes.

• Heavy Chain Segments: V (Variable), D (Diversity), J (Joining), and C (Constant).

• Light Chain Segments: V, J, and C (Note: Light chains lack the "D" segment).

Describe the two-step DNA excision process that occurs when forming a functional Heavy Chain gene in a lymphocyte.

1. D-J Joining: DNA excision randomly removes segments to bring one D segment adjacent to one J segment.

2. V-DJ Joining: A second random excision brings one V segment adjacent to the newly formed DJ unit.

• Result: A unique, rearranged "Lymphocyte DNA" sequence (V2​D17​J5​ in the slide's example).

After DNA rearrangement, how is the final mRNA for an antibody heavy chain produced?

1. Transcription: The rearranged DNA is transcribed into pre-mRNA.

2. RNA Splicing: The remaining "extra" sequences between the VDJ segment and the C (Constant) segment are removed.

3. Result: Functional mRNA ready for translation into a heavy chain protein.

Why is a "one gene = one antibody" system inefficient for the human immune system?

Because we need to produce billions of different antibodies to fight diverse pathogens. If each required its own gene, our genome would have to be impossibly large.

• Example from slide: 15 genes would only equal 15 different proteins.

How does "mixing and matching" gene segments increase antibody diversity?

By breaking a single gene into multiple segments (V, D, and J) that can be randomly combined, the number of possible proteins increases multiplicatively rather than additively.

Using the "Power of Combinations" logic, how do humans achieve roughly 1011 (100 billion) different antibody possibilities?

Through three levels of diversity:

1. Combinatorial: Randomly picking 1 V, 1 D, and 1 J segment.

2. Junctional: Adding/subtracting random nucleotides at the "joints" where segments meet.

3. Chain Pairing: Combining different Heavy chains with different Light chains.

Chromatin-mediated Regulation

• Condensed chromatin: gene silenced

  • Heterochromatin regions (centromere, telomere) contain few active genes

• Decondensed chromatin allows for gene expression

DNA Methylation generally leads to

chromatin condensation

DNA Methylation

Addition of methyl groups to selected cytosine in DNA

• Carried out by enzymes called DNA methylase/DNA

  methyl-transferase

CpG Island

a region of DNA with a high frequency of cytosine-guanine dinucleotides that are often located near gene promoters, influencing gene expression.

Methylation of lysine 4 of histone H3

is associated with gene activation and transcriptional regulation.

methylation of lysine 9 and 27

is linked to gene repression and chromatin condensation.

Acetylation

accomplished by the enzyme histone acetyl transferase (HAT) and generally promotes chromatin decondensation

Histone deacetylase (HDAC)

removes acetyl groups from histones

Chromatin remodeling proteins (SWI/SNF)

use the energy of ATP hydrolysis to slide nucleosomes or cause their ejection from a region of chromatin

Contrast the effects of DNA methylation and Histone acetylation on chromatin structure and gene expression.

• DNA Methylation: Leads to tighter chromatin packing → Gene Suppression (Off).

• Histone Acetylation: Leads to looser chromatin packing → Gene Activation (On).

Why is transcriptional regulation considered the "major regulation step" in gene expression?

It results in differential gene transcription, meaning the cell chooses which specific genes to turn into mRNA. This ensures different cell types (like a neuron vs. a muscle cell) produce the specific set of proteins they need.

What are regulatory transcription factors, and what are the two main types?

They are proteins that interact with DNA to control the rate of transcription.

1. Transcriptional Activators: Increase/promote transcription.

2. Transcriptional Repressors: Decrease/block transcription.

In the context of transcriptional regulation, what are control elements and their two primary forms?

They are specific DNA sequences that act as binding sites for transcription factors.

1. Enhancers: DNA sequences that bind activators (stimulate transcription).

2. Silencers: DNA sequences that bind repressors (inhibit transcription).

Can an Enhancer (E) function without a Promoter (P)?

No.

• Without a promoter, there is no transcription (Relative level = -).

• The promoter is required for the basal level of transcription (+); the enhancer simply "cranks up" that existing level (++++).

What are the three ways an enhancer's position/orientation can vary while still successfully activating transcription?

1. Distance: It can be close (-200) or very far (-1000) from the promoter. 2. Orientation: It works whether it's facing "forward" or "backward" (inverted). 3. Location: It can be upstream (before) or downstream (after) the gene.

How can an enhancer affect a promoter that is thousands of base pairs away?

Through DNA bending/looping.

• Activator proteins bind to the enhancer, and the DNA folds over so the enhancer can physically interact with the proteins at the promoter site.

What is the role of the Mediator complex in enhancer action?

It acts as a "bridge." It sits on the promoter and binds to the distant activators (on the enhancer), triggering the final assembly of RNA Polymerase and general transcription factors to start mRNA synthesis.