Large Ribosomal Subunit of Haloarcula

The Large Ribosomal Subunit (1s72), resolution 2.4Å ().

·· ·· ·· ··
·· ··

The Haloarcula Large Ribosomal Subunit
The molecular machine catalyzing petide bond synthesis is a ribozyme.

Introduction

The ribosome is a complex composed of RNA and protein that adds up to several million daltons in size and plays a critical role in the process of decoding the genetic information stored in the genome into protein as outlined in what is now known as the Central Dogma of Molecular Biology^[1]. Specifically, the ribosome carries out the process of translation, decoding the genetic information encoded in messenger RNA, one amino acid at a time, into newly synthesized polypeptide chains. The ribosome functions as a complex of two complexes of many proteins and RNAs of substantial length; these two complexes are the small ribosomal subunit and the large ribosomal subunit. The formation of peptide bonds occurs in the large subunit where the acceptor-stems of the tRNAs are docked.

In 2000, the large ribosomal subunit from Haloracula marismortui was solved. Haloracula is a halophilic archaea. The structure revealed that surprisingly no protein was observed close enough the site of peptide bond synthesis to be be involved in the chemistry of the peptidyl transferase reaction, meaning that RNA was responsible for catalysis and that the large subunit is a ribozyme. The structure also revealed the details of the tunnel which the nascent peptide chain would exit the ribosome.

Thomas Steitz shares a 2009 Nobel Prize for The Haloarcula Large Ribosomal Subunit Structure

For this landmark structure, Thomas A. Steitz of Yale University shared the 2009 Nobel Prize in Chemistry^[2][3] along with two other structural biologists working on the ribosome. It is important to note that the Steitz lab worked with the Moore lab on this phenomenal accomplishment although the Nobel committee limits the award itself to up to three laureates. This structure ranks among the known structures with highest impact.

Haloracula Large Ribosomal Subunit Components

The large subunit of the Haloracula marismortui ribosome sediments at 50S, as do the large subunits of archaea and eubacteria. It is composed of two chains of RNA, a 23S chain (2,922 nucleotides long, 946 kDa) and a 5S chain (122 bases long, 39 kDa). Assembled with the RNA are 27 protein chains (of a total of 31 known), varying in length from 49 (L39E, 6 kDa) to 337 amino acids (L3, 37 kDa)^[4].

The Haloarcula Large Ribosomal Subunit Structure in detail

The Haloarcula large ribosomal subunit at first glance:

.

• With no significant portion of the 50S subunit appearing topologically separate or capable of forming a stable structure on its own, the solved structure of the 1.6-million Dalton large subunit is one massive domain.
• On the other hand, the large subunit's partner in translation, the small subunit (30S), clearly has three domains.
• It is important to note that two stalks (the L1 stalk and L7/L12 stalk) seen in lower resolution structures on each side of the large ribosomal subunit at lower resolution^[5] are not visible in the higher resolution structure viewed here. Thus, in actuality the Haloarcula large subunit has a less monolithic appearance with other protuberances on either side of the central one, yet clearly not possessing the distinct domains formed by the distinct rRNA domains visible in the secondary structure (see below).

is a ribonucleoprotein macromolecule.

• This macromolecule is mostly RNA sprinkled with several proteins.
• 27 of the 31 known large subunit proteins are visible in the crystal structure. L1, which would be at the 'L1 stalk', is one of the proteins not seen.

two RNA chains:

1. a small 5S rRNA (122 nucleotides)which forms part of the central protuberance seen in the large subunit.
2. a large 23S rRNA (3045 nucleotides) - 2833 of the 3045 nucleotides of the 23S rRNA are seen in the structure.

The rRNA domains:

The secondary structure map of Haloarcula 23S rRNA (below) clearly shows six large RNA domains extending off a large major loop.

:

• (shown in blue)
• (shown in cyan)
• (shown in yellow)
• (shown in green)

• Helix 69 is a portion of Domain IV ^[6]and is part of one of the important conserved intersubunit bridges of the ribosome (b2a), although it is not visible in this structure. (The 13-nt stem-loop not seen would connect the highlighted spheres .) See the ribosome to see a structure where helix 69 is observed in the solved structure, extending from the large subunit under the A- and P- site tRNAs in the 70S ribosome and contacting the tRNAs and the small subunit decoding center. Helix 69 plays a roles in initiation, termination, and disassembly of the ribosome post-termination^[7][8][9].

• (shown in red)

• Domain V lies at the core of the subunit.   It is known to be intimately associated with the peptidyl transferase reaction that occurs during translation. This is further explored further below.

• (shown in purple)
• (shown in magenta), though a separate molecule, is effectively the seventh RNA domain of the large subunit.

The proteins:

• Proteins are generally globular. However, while about half the 27 proteins seen in the crystal structure of the large ribosomal subunit are globular (shown as orange), interestingly, the other half are extended or have large extended regions emanating from globular domains (shown as cyan).
• These extended proteins and regions are reminiscent of the intrinsically unfolded proteins that play roles in many other processes.

Zooming in to see some examples in more detail:

• L2, L15, and L39e illustrate proteins with extended regions.
• For contrast, the globular L7ae is shown. The extended regions are highlighted in cyan.
• L39e is extended over its entire length.

• View of L2, L15, L39e and L7ae again but with the RNA backbone shown. The extended regions are again highlighted in cyan.
• Using the mouse to spin around the structure clearly shows that the extended proteins penetrate into the interior to fill gaps between RNA secondary structure elements.
• The globular domains are the portions of the proteins on the subunit's exterior, nestling in the gaps and crevices of the folded RNA. You may need to use the mouse to move the structure around to convince yourself.
• This view is also a good point to note the fact that the ribosomal proteins do not encase the nucleic acid as with spherical viruses or with Tobacco mosaic virus, nor do the proteins become surrounded by the nucleic acid as in the nucleosome.

The ribosome is a ribozyme - protein DOES NOT participate directly in the chemistry of peptide bond synthesis:

(shown in red)

• In addition to unliganded subunit, the large subunit structure has been solved with substrate analogs which provides a detailed view of the direct role domain V plays in the chemistry of peptide bond synthesis. One of the analogs was the Yarus analog (CCA-puromycin), known to inhibit translation because it mimics normal substrate, specifically it resembles an unstable transition state intermediate formed during peptide bond synthesis and involving the extreme ends of the A- and P- tRNAs and atoms corresponding to parts of an amino acid. .

• with the Yarus analog.
• ^[10], and A2486 (E. coli #2451) of Domain V approaches this critical atom of the Yarus analog. The individual components of the Yarus analog are labeled.
• It is important to note that the ribosome is highly conserved, particularly the nucleotides close to the Yarus analog in Domain V and thus the major conclusions reached from the structure are applicable to all ribosomes.

• L2, L3, L4 and L10e are the nearest proteins to the Yarus analog bound to domain V of the subunit. Note that it may help to start from before hitting the above to get a good sense of these elements in the core of the subunit or use . With
• Examining the distance of the proteins from the phosphorous analog of the tetrahedral carbon (in yellow) indicates none of the proteins are close enough to be involved in the chemistry of peptide bond synthesis. Even the closest protein is over 15 Å away.(In the eubacterial ribosomes , e.g., 2j01,2i2v, and 2wdn, the N-terminus of a non-universally conserved protein, L27, also comes close to the active site^[11][12][13].)
• As touched on earlier in this section as well as in an earlier section, it is in fact, , leaving little doubt that the ribosome is indeed a ribozyme.

• Keep in mind that in order to make this large molecule load in reasonable times over the internet, the RNA has been simplified by leaving out the information for most of the bases and in fact if all the bases were included it would look even more crowded with RNA at the active site.

SUMMARY: Only RNA is in proximity to the site of peptide bond synthesis, and therefore the chemistry of peptide bond synthesis is not catalyzed by protein and in fact the ribosome is a ribozyme with the peptidyl transferase reaction being catalyzed by RNA.

Polypeptide Exit Tunnel:

• As the nascent chain grows, it advances into a tunnel about 100 angstroms long that passes through the large subunit, called the polypeptide exit tunnel.
• The diameter averages about 15Å; at no point is the diameter within the tunnel big enough to accommodate any folding of the nascent chain to a greater extent than alpha-helices^[14].
• The wall of the tunnel is mostly RNA (ca. 80%) and the proteins are shown translucently to emphasize this point and for clarity.
• , making a , and have been suggested to play roles in regulation of translation^{[15][16][17][18][19]}, particularly in the case of L22, although an involved portion is dispensable for growth^[20].
• encircle this opening which is where the nascent polypeptide chain will first emerge from the ribosome. Here the nascent chain can contact chaperones, such as trigger factor, and the machinery for transferring the growing chain across a membrane, the signal-recognition particle^[21][22].

• Each amino acid added to the growing peptide chain will begin its journey through the tunnel at this end.
• The in the active site. This makes sense since the analog represents the intermediate in peptide bond synthesis.
• A number of antibiotics, including the macrolide Azithromycin (zithromax), binds to this region of the tunnel.

Structures

Steitz and Moore labs original atomic-resolution structures^[23][24]: Haloarcula marismortui large ribosomal subunit - 1ffk and later refined to give 1jj2^[25], and then refined to give 1s72^[26], 2qa4^[27], and later 3cc2^[28]. Related: 1ffz, 1fg0. Assembled with the ribosomal RNAs (2,922 and 122 nucleotides long) in the structure are 27 protein chains (of a total of 31 known), varying in length from 49 (L39E, 6 kDa) to 337 amino acids (L3, 37 kDa).^[29] Specifically used on this page were 1s72 with Yarus analog interacting nucleotides from 1ffz.

See Also

• Ribosome
• Interactions between Antibiotics and the Ribosome
• Azithromycin bound to the Large Ribosomal Subunit of Haloarcula marismortui
• Small Ribosomal Subunit
• Nobel Prizes for 3D Molecular Structure
• Extremophiles
• Highest impact structures of all time
• Ribozyme
• 1nkw – The Large Ribosomal Subunit From Deinococcus radiodurans
• Kink-turn motif – analysis of the Haloarcula large ribosomal subunit revealed a common RNA motif. The location of the kink-turns in the Haloarcula large ribosomal subunit are shown on this page.
• For additional information, see: Bacterial Infections
• For additional information, see: Translation