Gene expression

State two structural differences between DNA and RNA, and explain which bases pair up during transcription when the template strand shows an adenine or a thymine.

To translate the $61$ sense codons in the standard genetic code, most eukaryotic cells only need to synthesize between $30$ and $45$ different tRNA species, instead of $61$.

1. a) Explain the thermodynamic and structural basis of Crick's 'wobble hypothesis' and justify why this spatial flexibility is restricted to the third nucleotide of the codon ($3'$ end) and the first of the anticodon ($5'$ end).
2. b) The amino acid valine is coded by four codons: $5'\text{-GUU-}3'$, $5'\text{-GUC-}3'$, $5'\text{-GUA-}3'$, and $5'\text{-GUG-}3'$. If a specific tRNA has inosine (I) at the $5'$ end of its anticodon (knowing that inosine can pair with U, C, and A), determine how many and which of these codons this single tRNA will be able to read, writing the sequence of said anticodon in the $5'\to 3'$ direction.
3. c) Reason the adaptive advantage for the organism of having the degeneracy of the genetic code and wobble concentrated at the third position of the codon, compared to them occurring at the first or second base when point mutations occur.

In gene therapy for Leber's hereditary optic neuropathy, researchers are investigating transferring a wild-type copy of a mitochondrial gene to the cell nucleus (allotopic expression) so that the protein is translated in the cytosol and imported into the mitochondrion. However, in the human mitochondrial genetic code, the triplet UGA codes for tryptophan, whereas in the standard nuclear code it acts as a termination codon.

1. a) Predict what would happen if the mitochondrial gene is transferred to the nucleus without modifying its sequence and is transcribed and translated on cytoplasmic ribosomes. What impact would this have on the length and functionality of the resulting polypeptide?
2. b) Design the site-directed mutagenesis strategy that the biotechnology team must apply to the coding sequence of the transgene before integrating it into the nucleus to ensure the synthesis of the native protein.
3. c) Justify why the mitochondrial genome (with only $37$ genes and a very small number of tRNAs) could 'afford' drift and changes in codon assignments throughout evolution, whereas in the nuclear genome this type of alteration is lethal to the cell.

Define what a gene is from the point of view of gene expression and state which two types of functional products a gene can generate when it is expressed.

In the human genome, the fibronectin gene contains numerous exons and introns. In fibroblasts, it gives rise to an insoluble protein isoform that forms part of the extracellular matrix, whereas in hepatocytes it originates a soluble isoform that circulates in the blood plasma.

1. a) Justify the molecular mechanism by which a single genomic DNA sequence can give rise to these two proteins with such different functions and locations.
2. b) A patient presents a point mutation consisting of a transition (G to A) at the splicing acceptor site of intron 4 of this gene. Predict two possible alterations in the structure of the resulting mature mRNA and their consequences for the synthesized protein.
3. c) If we clone this intact human gene (with all its introns and exons) into a plasmid and introduce it into Escherichia coli, reason whether the bacterium will synthesize functional human fibronectin.

A human gene of $24\,000$ base pairs (bp) consisting of $8$ exons and $7$ introns is studied. After being completely processed, its mature mRNA in muscle cells is $1200$ nucleotides long (including the UTR regions and the poly-A tail).

1. a) If the template strand of genomic DNA is hybridized in the laboratory with said mature muscle mRNA and the resulting heteroduplex is observed under an electron microscope, predict what morphology it will present and how many single-stranded loops will be observed at most. Justify which nucleic acid each loop will be made of.
2. b) A student claims that, having $1200$ nucleotides, the resulting protein will be exactly $400$ amino acids long. Refute this statement with reasoning, providing at least two arguments of a molecular nature.
3. c) In nervous tissue, a mature mRNA originating from the same gene but with a size of $1500$ nucleotides is isolated. Reason what phenomenon has occurred and predict whether the neuronal protein will necessarily be longer than the muscular one, justifying your answer.

Streptomycin is a bactericidal antibiotic that binds to the $30\text{S}$ subunit of the prokaryotic ribosome ($70\text{S}$), causing mRNA misreading and inhibiting protein synthesis. Although it is considered selective against eukaryotic cytoplasmic ribosomes ($80\text{S}$), patients undergoing intensive treatments often experience nephrotoxicity and ototoxicity associated with cellular energy dysfunction.

1. a) Based on the endosymbiotic theory, justify why this drug can damage human cells and cause an ATP deficit despite the described target selectivity.
2. b) When analyzing a streptomycin-resistant bacterial strain, it is verified that it does not degrade the drug nor does it possess active efflux pumps, but it carries a mutation in the bacterial chromosome gene that encodes the $16\text{S}$ rRNA. Explain how this mutation confers resistance without completely impairing ribosome function.
3. c) In a cellular experiment, cycloheximide (an $80\text{S}$ ribosome inhibitor) and chloramphenicol (a $70\text{S}$ ribosome inhibitor) are used. Predict what will happen to the assembly of the multienzyme complexes of the mitochondrial electron transport chain (whose subunits have a dual genetic origin) in the presence of each of these compounds separately.

When observing gene expression in bacteria (Escherichia coli) by electron microscopy, 'Christmas tree' structures can be distinguished where multiple ribosomes translate mRNA while it is still being synthesized by RNA polymerase coupled to genomic DNA. In contrast, this is never observed in a human cell.

1. a) Predict what immediate consequence the addition of rifampicin (an inhibitor that blocks RNA polymerase initiation but not ribosome activity) will have on bacterial protein synthesis, and compare this effect with what would occur in the short term in the cytoplasm of a eukaryotic cell.
2. b) Reason why bacterial transcription-translation coupling is mechanically incompatible with the presence of introns and with the splicing mechanism typical of eukaryotes.
3. c) The gene regulation mechanism by 'attenuation' in the bacterial tryptophan operon depends on the ribosome's rate of progress through a leader sequence determining the formation of termination hairpins in the nascent RNA. Justify in a reasoned manner why this regulatory system is unfeasible in eukaryotic cells.

The following diagram represents a functional ribosome during the elongation phase of translation:

1. a) Justify and deduce the polarity ($5'$ and $3'$ ends) of the mRNA strand and the direction in which the ribosome will move along it.
2. b) Write the sequence of the anticodon of the tRNA marked with a question mark (?), mandatory specifying its $5'\to 3'$ polarity, and reason which amino acid it carries knowing that the GCU codon codes for alanine.
3. c) In a mutant strain, a suppressor tRNA is produced that is capable of recognizing the UAA codon by inserting the amino acid tyrosine. Predict what will happen to translation and to the length of the resulting protein when the ribosome reaches the position marked with the stop codon compared to the wild-type cell.