8.7: Getting more complex- gene regulation in eukaryotes — Read

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Diagram illustrating cellular processes, including mRNA export from the nucleus and interaction with ribosomes in the cytoplasm. We will briefly reiterate a few points on how gene expression and polypeptide synthesis differ between prokaryotes and eukaryotes. The first and most obvious difference in gene expression between prokaryotes and eukaryotes (us) is the presence of a nucleus, a distinct domain within the eukaryotic cell that separates the bulk of the cell’s genetic material, its DNA, from the cytoplasm, where the ribosomes are located (→). Aside from those within mitochondria and chloroplasts, the DNA molecules of eukaryotic cells are located within the nucleus. The barrier between nuclear interior and cytoplasm is known as the nuclear envelope. No similar barrier exists between DNA and ribosomes in prokaryotes. In both bacteria and archaea the DNA is in direct contact with the cytoplasm. In eukaryotes, a newly synthesized mRNA molecule undergoes a number of modifications before it comes into contact with the cytoplasm - it can be "spliced" and modified (processed) at both its 5’ and 3’ ends. Only after RNA processing has occurred will the “mature” mRNA be exported out of the nucleus , through a nuclear pore, and into the cytoplasm, where it can interact with ribosomes. Prokaryotic mRNAs are generally not processed.

The nuclear envelope complex, considered in greater detail in cell biology courses, consists of two lipid bilayer membranes punctuated by nuclear pores. These pores are macromolecular complexes (protein machines) of ~125,000,000 daltons. Molecules of less than ~40,000 daltons can generally 3D illustration of a molecular pore structure with colorful surrounding elements, labeled "pore" in the center. move passively through a nuclear pore; larger molecules must be actively transported through a process coupled to the a thermodynamically favorable reaction, in this case the hydrolysis of guanosine triphosphate (GTP). The movement of larger molecules into and out of the nucleus through nuclear pores is regulated by what are known as nuclear localization and nuclear export sequences, located within polypeptides. These sequences are recognized by proteins (receptors) associated with the pore complex (→). A protein with an active nuclear localization sequence (NLS) will be found primarily in the nucleus while a protein with an active nuclear exclusion sequence (NES) will be found primarily in the cytoplasm. By controlling NLS and NES activities, a protein can come to accumulate, in a regulated manner, in either the nucleus or the cytoplasm, or can be found in both cellular regions. As we will see later on, the nuclear envelope breaks down during cell division (mitosis) in many but not all eukaryotes. Tares in the nuclear envelope have been found to occur when migrating cells try to squeeze through small openings.^{^[344]} Once the integrity of the nuclear envelope is re-established. Proteins with NLS and NES sequences moved back to their appropriate locations through active, that is energy dependent, coupled reaction-based transport processes.

Nonsense mediated RNA decay

An originally surprising observation was that the truncated polypeptide generated by a non-sense mutation could produce phenotypic effects that were more severe than those associated with the failure to produce any polypeptide at all. To protect against the negative effects of non-sense mutations, particularly those that occur well "up-stream" of the normal stop codon, eukaryotic organisms have a defense mechanism known as non-sense mediated decay. In a typical gene, the "normal" stop codon is generally within an exon located near the 3’ end of the newly synthesized "pre-mRNA". During mRNA processing, introns are recognized and removed by the splicing system (↓); the 5' end is"capped" and the 3' end processed and (generally) a stretch of A nucleotides, a "polyA tail", is Diagram illustrating mRNA splicing, transport, ribosome loading, and translation processes in gene expression. added. Typically, all of these modifications are completed before the processed transcript, now an mRNA, is transported through the nuclear pore complex into the cytoplasm.

The removal of an intron leads to the formation of an exon-exon junction and the association of an exon-exon junction protein complex (EJC) "upstream" of each exon-exon junction (↓).^{^[345]} When a ribosome engages with the 5' end of the mRNA and moves down the mRNA during translation it displaces the EJCs, so when the first ribosome reaches the end of the mRNA's coding region all of the EJCs have been removed. The stability of the EJC-free mRNA is regulated by signals located primarily in its 5’ and 3’ untranslated regions.

The situation is different when a non-sense mutation generates a stop codon within an upstream exon ("mutant mRNA" →). The ribosome engages with the mRNA and continues until it reaches this stop codon, upon which release factor binds and the ribosome disengages. The EJCs downstream of the mutation-generated stop codon remain associated with the mRNA. The failure to remove the EJCs marks the mRNA as aberrant and triggers the non-sense mediated decay response.^{^[346]} Non-sense mediated decay activation leads to the degradation of mRNAs containing out-of-context non-sense codons and dramatically reduces the synthesis of potentially toxic polypeptides. In a further weird twist, it has been reported that RNA fragments generated from the degraded mRNA can re-enter the nucleus and regulate other genes - which further complicate the already complicated molecular relationship between mutation, genotype, and phenotype.^{^[347]}

Alarm generation: another secondary effect of disrupting gene expression

The translation system is a major consumer of energy within the cell.^{^[348]} When a cell is starving, it does not have the energy to generate amino acid charged tRNAs. The result is that uncharged tRNAs Illustration showing ribosomal structure with tRNAs, highlighting how uncharged tRNAs accumulate when cells starve. accumulate (→). Since uncharged tRNAs can fit into the amino-acyl-tRNA binding sites on the ribosome, their presence increases the probability of unproductive tRNA interactions with the mRNA-ribosome complex, a situation that can lead to the premature termination of translation. When this occurs the stalled ribosome generates a signal that can lead to adaptive changes in the cell that enable the cell to survive for long periods in a “dormant” state.^{^[349]}

Another response that can occur is a more social one. Some cells in the population can “sacrifice” themselves for their closely related neighbors, remember kin selection and inclusive fitness. By shutting down transcription and translation, an addiction module can undergo programmed cell death. That mechanism is based on a toxin and anti-toxin system that differ in their stability. Just as ribonucleases degrade mRNAs, proteases degrade proteins and polypeptides. How stable a protein/polypeptide is depends upon its structure, which we will consider soon, and the presence of proteases that degrade it. Interrupting protein synthesis leads to the rapid disappearance (turn-over) of the anti-toxin while the toxin persists, leading to cell death, which in turn leads to the release of the cell’s nutrients, nutrients that can be used by its neighbors, in part to maintain active gene expression and protein synthesis. Of course, sacrificing for ones neighbors makes evolutionary sense only if one has neighbors and those neighbors are close relatives.

Questions to answer:

A gene has many introns - provide a model for how it might encode multiple functionally distinct polypeptides.
How can a mutation in splice site sequence influence gene expression and protein function?
How does non-sense mediated decay (NMD) protect against potentially deleterious mutations (alleles)?
Why would a cell want to stop (rather than continue) polypeptide synthesis when it is starving?