Deep Inside the Ribosome Factory

Hundreds of processes are involved in cranking out ribosomes. One is carried out by an RNA helicase.

Our evolutionarily older molecules have been through a lot, and show the scars of billions of years of jury-rigging. The ribosome is among the oldest, and most ornately decorated, with dozens of extra proteins pasted around the outside, chemical modifications of its RNA and proteins, and a system of scaffolding and maturation factors. It even has its own organelle to develop in- the nucleolus. The nucleolus organizes spontaneously around the portions of the genome that encode the ribosomal RNA (rRNA), which are transcribed in prodigious amounts and whose products go through a lengthy maturation process.

To give an idea of the scale of all this, the ribosomal RNA is about five thousand nucleotides long, and about a hundred of these nucleotides are chemically altered by extra processes, all of which are highly unsual, at least versus normal messenger RNAs. At least three sites are cleaved during maturation, and seventy nine different proteins are added that join the mature structure. There are also over two hundred accessory proteins and seventy-six small RNAs that do not join the mature ribosome, but are needed to facilitate the various folding and chemical modifications during the construction process, which is all done in an ordered fashion. In a cell like yeast, two thousand ribosomes are assembled per minute, taking up a huge share of cellular resources. For example, the ribosomal proteins take up about sixty percent of the mRNA production machinery in a growing yeast cell.

Overview of ribosomal RNA maturation. 25S is the large subunit precursor rRNA segment, while 18S is the small subunit precursor. The ITS segments are intervening  portions that are clipped out of the original long RNA. The small subunit (orange) has a somewhat quicker maturation path than the large subunit (red). Shapes change extensively as the nascent RNAs get prodded and pulled into their final shapes, as if the nucleolus were a tiny little hair salon.

The two halves of the ribosome, the small and large subunits, are separately made and matured, (with all the various constituent and helper proteins being imported back from the cytoplasm to take up their places in these nascent structures) and then exported from the nucleolus out to nucleus and on to the cytoplasm, where some final maturation steps take place, including removal of any remaining accessory factors, Last comes a test run through a fake synthesis cycle without any mRNA or tRNA substrates, after which defective ribosomes are destroyed.

This system is truly daunting in its complexity, but obviously not complexity borne of design. Rather, it is borne of desperation, as bandaid after bandaid has been applied to produce the massive machine that currently sits at the heart of protein synthesis. It is a classic snowball effect, where items added to provide a modicum of extra stability, speed, or accuracy each reinforce the conservation of the core mechanism, making it increasingly impossible to create any radical change or redesign. Optimization in this case has been the enemy of efficiency, since the core of the enzyme, based on RNA, is so intrinsically inefficient.

Structure of the ribosome at an intermediate state, when helicase Spb4 (pink) is attached. All of the colored proteins, in fact, are modifier/accessory factors and are destined to fall off eventually. The ITS2 is the intervening sequence from the ribosomal RNA which has also not yet been cleaved and prised off the structure, but will be soon.

A recent paper sought to look at one small part of this byzantine construction process- where a helicase attaches and participates in one of the later steps as the nascent large ribosomal subunit exits from the nucleolus to the nucleoplasm. Helicases are enzymes that help nucleic acids unwind, (and rewind), which is just the kind of thing the ribosomal RNA so desperately needs as it wends its way from a linear RNA to the compact final structure. The authors use the new method of cryo-electron microscopy to obtain atomic structures of the large subunit in various stages of dress. One image, below, shows some detail about how helicase SPB4 (pink) holds on to one small segment of the ribosomal RNA, wrenches it apart, and thus enables its small structural transition.

Closer structure of Spb4, showing how it grips the ribosomal RNA, which is denoted by the high numbers, G1919 to G1948, based on the nucleotide positions. It is also an ATPase, which powers its helicase activities. RecA1 and RecA2 refer to proteins domains within Spb4 that are characteristic of helicase enzymes, as their "hands". CTD refers to the end of the protein, its carboxy-terminal domain.

The paper is a long-winded discussion of the many protein-protein contacts being made among these accessory factors,  which come on first, then next, then which force others off, etc. Their conclusions are shown below, as a sequence of states where, though at first glance nothing seems to have happened, the final state is quite different in detail from the state C coming in, not only in terms of the accessory proteins present, but also in the structure of the core ribosome. Only eight different proteins are in play here, so this is a tiny slice of the whole process. What is happening to the ribosomal RNA, the target of all this activity? They provide a rundown of some of Spb24's effects as follows, though a full appreciation of its role remains unclear:

The accommodation of the rRNA substrate between the two RecA-like domains induces bending and strand separation of the rRNA around the base of ES27, resulting in an alternate base-pairing of helices H62/H63/H63a compared to nucleoplasmic maturation intermediates and mature 60S subunits. This may explain why the rRNA area at the base of 25S domain IV initially appears to form stable duplexes, while it becomes more flexible and accessible for chemical modification in presence of Spb4, suggesting that the helicase disrupts this region upon its association. In addition to the catalytic domain, Spb4’s essential CTD appears significantly involved in inducing substrate RNA strand disruption and establishing this alternate conformation. In the obtained substrate- bound state, the first half of the CTD (aa 406-499) is tightly docked onto the C-terminal RecA- like domain (RecA2) and binds H62/H63 nucleotides A1936 to C1941, thereby maintaining separation of the rRNA strands. Furthermore, a conserved tryptophan (W536) within the flexible C-terminal tail of the CTD (aa 500-606) intercalates between nucleotides of the immature H62/H63/H63a rRNA, which later adopts its mature-like fold in nucleoplasmic pre-60S particles. - Authors; (ES27 denotes a region of the ribosomal RNA near the active site, as does domain IV. H62/63 denote helices of rRNA, as shown in the diagram above.)

 

Schematic of what is happening to the large ribosomal subunit during these few steps. Accessory factors by the dozens are coming and falling off as the whole process happens, while also guiding the ribosome through its transport process from nucleolus out to the cytoplasm, while in addition doing various QC steps that can shunt defective complexes to cellular waste bins.

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