At this point, we will not take very much time to go into how gene expression in particular, and polypeptide synthesis in general differ between prokaryotes and eukaryotes except to point out a few of the major differences, some of which we will return to, but most will be relevant only in more specialized courses. The first and most obvious difference is the presence of a nucleus, a distinct domain within the eukaryotic cell that separates the cell’s genetic material, its DNA, from the cytoplasm. The barrier between nuclear interior and cytoplasm is known as the nuclear envelope (no such barrier exists in prokaryotic cells, the DNA is in direct contact with the cytoplasm.) The nuclear envelope consists of two lipid bilayer membranes punctuated by nuclear pores, macromolecular complexes (protein machines). While molecules of molecular weight less than ~40,000 daltons can generally pass through the nuclear pore, larger molecules must be actively transported, that is, in a process that is coupled to a thermodynamically favorable reaction, in this case the hydrolysis of guanosine triphosphate (GTP) rather than ATP. 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 withinin polypeptides. These are recognized by proteins (receptors) associated with the pore complex. A protein with an active nuclear localization sequence (NLS) will be found in the nucleus while a protein with an active nuclear exclusion sequence (NES) will be in the cytoplasm. By controlling NLS and NES activity a protein can come to accumulate in a regulated manner in either the nucleus or the cytoplasm. .As we will see later on the nuclear envelope breakdowns occurs during cell division (mitosis) in many but not all eukaryotes. Tears in the nuclear envelope have also been been found to occur when migrating cells trying to squeeze through small openings243. Once the integrity of the nuclear envelop is re-established, proteins with NLS and NES sequences move back to their appropriate location within the cell.
Aside from those within mitochondria and chloroplasts, the DNA molecules of eukaryotic cells are located within the nucleus. One difference between eukaryotic and bacterial genes is that the transcribed region of eukaryotic genes often contains what are known as intervening sequences or introns; introns involve sequences that do not encode a polypeptide. After an RNA is synthesized introns are removed enzymatically, resulting in a shorter mRNA. As a point of interest, which sequences are removed can be regulated, this can produce multiple different mRNAs from the same gene, mRNAs that encode somewhat (and often functionally) different polypeptides. In addition to removing introns, the mRNA is further modified (processed) at both its 5’ and 3’ ends. Only after RNA processing has occurred is the now “mature” mRNA exported out of the nucleus, through a nuclear pore, into the cytoplas where it can interact with ribosomes. One further difference from bacteria is that the interaction between a mature mRNA and the small ribosomal subunit involves the formation of a complex in which the 5’ and 3’ ends of the mRNA are brought together into a circle. The important point here is that unlike the situation in bacteria, where mRNA is synthesized into the cytoplasm and so can immediately interact with ribosomes and begin translation (even before the synthesis of the RNA is finished) transcription and translation are distinct processes in eukaryotes.This makes the generation of multiple, functionally distinct RNAs (through mRNA processing) from a single gene possible and leads to significantly greater complexity from only a relatively small increase in the number of genes.