Gene Expression

Which enzyme catalyses the transcription of DNA into RNA? State on which of the two strands of the gene it acts and in which direction it synthesises the RNA molecule.

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.

From the DNA template strand 3'-TACGGAACT-5', obtain the resulting mRNA sequence indicating its polarity, and state the maximum number of amino acids this fragment can encode.

What is a codon? State how many nucleotides make it up, how many distinct codons exist, and how many different amino acids they encode in total.

Look at the diagram of the flow of genetic information. State which molecules are labelled A, B and C, which processes occur at arrows 1 and 2, and what the area surrounded by the dashed line represents.

Consider the following fragment of the coding sequence of an mRNA that is translated into a cytoplasmic protein:

$5'\text{--- AUG - UCU - UCC - UCA - UCG --- } 3'$

(Note: the four codons following the AUG start codon all code for the amino acid serine).

1. a) Based on this specific case, justify why the genetic code is degenerate and demonstrate that this property is not equivalent to saying that the code is "ambiguous".
2. b) Suppose that two independent point mutations are introduced: Mutant 1 changes the second codon from UCU to UCC; Mutant 2 changes the third codon from UCC to UAC (which codes for tyrosine). Compare the impact of both mutations on the amino acid sequence of the protein and classify each mutation according to its functional effect.
3. c) Reason what adaptive advantage is conferred by the fact that the degeneracy of the genetic code is almost always concentrated at the third base of each triplet (wobble hypothesis) rather than at the first or second, in the face of spontaneous point mutations.

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 transcription and translation and state where in the eukaryotic cell each of these processes takes place.

What is the start codon of translation and which amino acid does it encode? Explain what function the stop codons fulfil and what happens when the ribosome reaches one of them.

Briefly describe what the ribosome does during translation: how it reads the mRNA, how the amino acids arrive and what type of bond is formed between them.

Based on the following genetic code table:

1. a) Translate the following $\text{mRNA}$: $5'\text{-AUGGCUUAA-}3'$, and indicate the resulting amino acid sequence.
2. b) Explain what role the first and the last codon of the fragment play.

During translation, the mRNA is read in consecutive codons, with no separators between them. Explain what the reading frame is and reason what would happen to the synthesised protein if the ribosome started reading one nucleotide before the correct position.

Differentiate the functions of mRNA, tRNA and rRNA in protein synthesis, indicating what each type of RNA contributes to the process.

Describe the characteristic structure of a tRNA (clover-leaf shape, anticodon and 3' end) and explain how the cell manages to get each tRNA loaded with its correct amino acid, mentioning the enzymes responsible.

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.

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.

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.

Order the following stages of gene expression in a eukaryotic cell and justify the chosen order: binding of the mRNA to the ribosome, mRNA processing, translation in the cytosol, exit of the mRNA from the nucleus, transcription in the nucleus.

A protein is made up of $300$ amino acids. a) Calculate how many nucleotides the mature mRNA that encodes it has, at a minimum, including the stop codon. b) Calculate how many base pairs the gene would occupy in an organism without introns. c) Reason why in eukaryotic organisms the gene is usually much longer than the corresponding mature mRNA.

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.