Large Ribosomal Subunit of Haloarcula

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This page, as it appeared on August 1, 2010, was featured in this article in the journal Biochemistry and Molecular Biology Education.



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



Contents

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

 
Drag the structure with the mouse to rotate
The Large Ribosomal Subunit (1s72), resolution 2.4Å ().
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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.

 
Drag the structure with the mouse to rotate
The Large Ribosomal Subunit (1s72), resolution 2.4Å ().
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:
  • (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:

 
Drag the structure with the mouse to rotate
The Large Ribosomal Subunit (1s72), resolution 2.4Å ().
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  • 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:

 
Drag the structure with the mouse to rotate
The Large Ribosomal Subunit (1s72), resolution 2.4Å ().
·· ·· ·· ··
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(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., 4v51,4v50, and 4v5d, 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:

 
Drag the structure with the mouse to rotate
The Large Ribosomal Subunit (1s72), resolution 2.4Å ().
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  • 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.
In 2013, the structure was re-refined to give 4v9f[30] These models with some additional portions observed have not been incorporated into this page.

See Also

References and Notes

  1. ↑ the Central Dogma of Molecular Biology clarified
  2. ↑ The Nobel Prize in Chemistry 2009 page at The Official Web Site of the Nobel Prize
  3. ↑ Steitz TA. From the structure and function of the ribosome to new antibiotics (Nobel Lecture). Angew Chem Int Ed Engl. 2010 Jun 14;49(26):4381-98. PMID:20509130 doi:10.1002/anie.201000708
  4. ↑ Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905-20. PMID:10937989
  5. ↑ Ban N, Freeborn B, Nissen P, Penczek P, Grassucci RA, Sweet R, Frank J, Moore PB, Steitz TA. A 9 A resolution X-ray crystallographic map of the large ribosomal subunit. Cell. 1998 Jun 26;93(7):1105-15. PMID:9657144
  6. ↑ Helix 69 is shown clearly in Domain IV in a detailed secondary structure of Haloarcula marismortui 23S rRNA
  7. ↑ Gao N, Zavialov AV, Ehrenberg M, Frank J. Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits. J Mol Biol. 2007 Dec 14;374(5):1345-58. Epub 2007 Oct 16. PMID:17996252 doi:10.1016/j.jmb.2007.10.021
  8. ↑ Kipper K, Hetenyi C, Sild S, Remme J, Liiv A. Ribosomal intersubunit bridge B2a is involved in factor-dependent translation initiation and translational processivity. J Mol Biol. 2009 Jan 16;385(2):405-22. Epub 2008 Nov 5. PMID:19007789 doi:10.1016/j.jmb.2008.10.065
  9. ↑ Ali IK, Lancaster L, Feinberg J, Joseph S, Noller HF. Deletion of a conserved, central ribosomal intersubunit RNA bridge. Mol Cell. 2006 Sep 15;23(6):865-74. PMID:16973438 doi:10.1016/j.molcel.2006.08.011
  10. ↑ Nissen P, Hansen J, Ban N, Moore PB, Steitz TA. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000 Aug 11;289(5481):920-30. PMID:10937990
  11. ↑ Maguire BA, Beniaminov AD, Ramu H, Mankin AS, Zimmermann RA. A protein component at the heart of an RNA machine: the importance of protein l27 for the function of the bacterial ribosome. Mol Cell. 2005 Nov 11;20(3):427-35. PMID:16285924 doi:10.1016/j.molcel.2005.09.009
  12. ↑ Trobro S, Aqvist J. Role of ribosomal protein L27 in peptidyl transfer. Biochemistry. 2008 Apr 29;47(17):4898-906. Epub 2008 Apr 8. PMID:18393533 doi:10.1021/bi8001874
  13. ↑ Voorhees RM, Weixlbaumer A, Loakes D, Kelley AC, Ramakrishnan V. Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome. Nat Struct Mol Biol. 2009 May;16(5):528-33. Epub 2009 Apr 12. PMID:19363482 doi:10.1038/nsmb.1577
  14. ↑ Voss NR, Gerstein M, Steitz TA, Moore PB. The geometry of the ribosomal polypeptide exit tunnel. J Mol Biol. 2006 Jul 21;360(4):893-906. Epub 2006 May 30. PMID:16784753 doi:10.1016/j.jmb.2006.05.023
  15. ↑ Nakatogawa H, Ito K. The ribosomal exit tunnel functions as a discriminating gate. Cell. 2002 Mar 8;108(5):629-36. PMID:11893334
  16. ↑ Berisio R, Schluenzen F, Harms J, Bashan A, Auerbach T, Baram D, Yonath A. Structural insight into the role of the ribosomal tunnel in cellular regulation. Nat Struct Biol. 2003 May;10(5):366-70. PMID:12665853 doi:10.1038/nsb915
  17. ↑ Gabashvili IS, Gregory ST, Valle M, Grassucci R, Worbs M, Wahl MC, Dahlberg AE, Frank J. The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. Mol Cell. 2001 Jul;8(1):181-8. PMID:11511371
  18. ↑ Lawrence MG, Lindahl L, Zengel JM. Effects on translation pausing of alterations in protein and RNA components of the ribosome exit tunnel. J Bacteriol. 2008 Sep;190(17):5862-9. Epub 2008 Jun 27. PMID:18586934 doi:10.1128/JB.00632-08
  19. ↑ Fulle S, Gohlke H. Statics of the ribosomal exit tunnel: implications for cotranslational peptide folding, elongation regulation, and antibiotics binding. J Mol Biol. 2009 Mar 27;387(2):502-17. Epub 2009 Jan 27. PMID:19356596 doi:10.1016/j.jmb.2009.01.037
  20. ↑ Zengel JM, Jerauld A, Walker A, Wahl MC, Lindahl L. The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control. RNA. 2003 Oct;9(10):1188-97. PMID:13130133
  21. ↑ Giglione C, Fieulaine S, Meinnel T. Cotranslational processing mechanisms: towards a dynamic 3D model. Trends Biochem Sci. 2009 Aug;34(8):417-26. Epub 2009 Jul 31. PMID:19647435 doi:10.1016/j.tibs.2009.04.003
  22. ↑ Pool MR. A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase. J Cell Biol. 2009 Jun 1;185(5):889-902. Epub 2009 May 25. PMID:19468070 doi:10.1083/jcb.200807066
  23. ↑ Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905-20. PMID:10937989
  24. ↑ Nissen P, Hansen J, Ban N, Moore PB, Steitz TA. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000 Aug 11;289(5481):920-30. PMID:10937990
  25. ↑ Klein DJ, Schmeing TM, Moore PB, Steitz TA. The kink-turn: a new RNA secondary structure motif. EMBO J. 2001 Aug 1;20(15):4214-21. PMID:11483524 doi:http://dx.doi.org/10.1093/emboj/20.15.4214
  26. ↑ Klein DJ, Moore PB, Steitz TA. The roles of ribosomal proteins in the structure assembly, and evolution of the large ribosomal subunit. J Mol Biol. 2004 Jun 25;340(1):141-77. PMID:15184028 doi:10.1016/j.jmb.2004.03.076
  27. ↑ Kavran JM, Steitz TA. Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements. J Mol Biol. 2007 Aug 24;371(4):1047-59. Epub 2007 Jun 4. PMID:17599351 doi:10.1016/j.jmb.2007.05.091
  28. ↑ Blaha G, Gurel G, Schroeder SJ, Moore PB, Steitz TA. Mutations outside the anisomycin-binding site can make ribosomes drug-resistant. J Mol Biol. 2008 Jun 6;379(3):505-19. Epub 2008 Apr 8. PMID:18455733 doi:http://dx.doi.org/10.1016/j.jmb.2008.03.075
  29. ↑ Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905-20. PMID:10937989
  30. ↑ Gabdulkhakov A, Nikonov S, Garber M. Revisiting the Haloarcula marismortui 50S ribosomal subunit model. Acta Crystallogr D Biol Crystallogr. 2013 Jun;69(Pt 6):997-1004. doi:, 10.1107/S0907444913004745. Epub 2013 May 11. PMID:23695244 doi:http://dx.doi.org/10.1107/S0907444913004745

Additional Literature

  • Moore PB. The ribosome returned. J Biol. 2009;8(1):8. Epub 2009 Jan 26. PMID:19222865 doi:10.1186/jbiol103
  • Steitz TA. A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol. 2008 Mar;9(3):242-53. PMID:18292779 doi:10.1038/nrm2352
  • Rodnina MV, Wintermeyer W. The ribosome goes Nobel. Trends Biochem Sci. 2010 Jan;35(1):1-5. Epub 2009 Dec 2. PMID:19962317 doi:10.1016/j.tibs.2009.11.003
  • Schmeing TM, Ramakrishnan V. What recent ribosome structures have revealed about the mechanism of translation. Nature. 2009 Oct 29;461(7268):1234-42. Epub 2009 Oct 18. PMID:19838167 doi:10.1038/nature08403
  • Ramakrishnan V, Moore PB. Atomic structures at last: the ribosome in 2000. Curr Opin Struct Biol. 2001 Apr;11(2):144-54. PMID:11297922
  • Schroeder KT, McPhee SA, Ouellet J, Lilley DM. A structural database for k-turn motifs in RNA. RNA. 2010 Aug;16(8):1463-8. Epub 2010 Jun 18. PMID:20562215 doi:10.1261/rna.2207910
  • Petrov AS, Bernier CR, Hershkovits E, Xue Y, Waterbury CC, Hsiao C, Stepanov VG, Gaucher EA, Grover MA, Harvey SC, Hud NV, Wartell RM, Fox GE, Williams LD. Secondary structure and domain architecture of the 23S and 5S rRNAs. Nucleic Acids Res. 2013 Jun 14. PMID:23771137 doi:10.1093/nar/gkt513

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