Archaeal Ribosomes

George Harauz, University of Guelph, Guelph, Canada Abdiwahab Musse, University of Guelph, Guelph, Canada
The protein synthesis machinery (ribosomes) of archeae, the third kingdom of life, have adapted to function under conditions of extreme salt and/or temperature, and show many features similar to their bacterial and eukaryotic counterparts.

Secondary article
Article Contents
. Introduction . Ribosomal Subunits . Ribosomal Proteins . Ribosomal RNA . Protein Synthesis and Targeting . Thermal and High Salt Stability

Introduction
The archaea (archaebacteria) are unique organisms and represent a new, third domain of life (Woese et al., 1978). Phylogenetically, the archaea comprise three distinct phenotypes: the methanogens, the halophiles and the sulfur-dependent thermophiles. Cytologically, these microorganisms are prokaryotes, in the sense that they are unicellular and have no membrane-bounded internal cytoplasmic compartments. However, they are more related to eukaryotes than bacteria in terms of their molecular and gene structures. Consequently, archaea provide an evolutionary link between the eukaryotes and the bacteria, and clues to understanding the nature of the ancestor common to all life. Moreover, the study of the archaeal cellular components and macromolecules is key to our understanding of the survival mechanism of the archaea in their sometimes extreme habitats. Ribosomes play an important role in phylogenetic studies and were pivotal in identifying the archaea as a distinct and a new kingdom of life. Their ribosomes possess properties that are unique to their kingdom, but also show eukaryotic- and bacterial-like features. As a result, archaeal ribosomes are considered to be evolutionary mosaic ribosomes. Here, we review the structural and functional components of archaeal ribosomes and some mechanistic aspects of the archaeal translational engine that allow archaea to secure sustainable life in such extreme environments as geothermal hot springs and in extremely high salt concentrations. We discuss the structures of archaeal large and small ribosomal subunits, and their constituent ribosomal proteins and ribonucleic acids (RNAs) (r-proteins and rRNAs, respectively). In specic references to archaeal, bacterial and eukaryotic macromolecules, the prexes a, e and u, respectively, are used.

. Conclusion

Ribosomal Subunits
The large (50S) and small (30S) subunits of archaeal ribosomes are similar in size to those of the bacterial ribosomes and are smaller than the corresponding (60S and 40S, respectively) eukaryotic ribosomal subunits. Mor-

phologically, the archaeal ribosomal subunits show both bacterial- and eukaryotic-like features. The large ribosomal subunit of the archaeal ribosome is made up of one molecule of 23S rRNA and 5S rRNA, plus approximately 30 r-proteins (Amils et al., 1993). Early electron microscopical studies of archaeal large ribosomal subunits (Henderson et al., 1984) revealed features resembling those present in bacterial large ribosomal subunits. For example, the archaeal large ribosomal subunit possesses a stalk that in bacteria has been shown to contain the ribosomal A protein domain (Figure 1) (Matheson, 1985). In bacteria, the ribosomal A protein domain contains four copies of r-proteins eL7/eL12 and one copy of r-protein eL10. The eL7/eL12eL10 protein complex binds to the 5- region of 23S rRNA via protein eL10, and this binding process is stimulated by another protein, eL11 (Matheson, 1985 and references therein). In archaea, the ribosomal A protein domain of the stalk contains four copies of aL20 and one copy of aL11. Sequence analyses of aL20 and aL11 have shown them to be equivalent to the Escherichia coli eL12 and eL11 (rather than eL10) r-proteins, respectively. The archaeal stalk region, like that of its bacterial counterpart, is presumed to be involved in the interaction and regulation of elongation factors during protein synthesis. Electron images of the archaeal large ribosomal subunit also show the presence of the central and L1 protuberances, features that are common to all kingdoms, albeit with slight dierences in appearance and protein composition. The sulfur-dependent archaea were once suggested to represent a new, fourth, kingdom, called eocytes, on the basis of perceived (in electron micrographs) features such as the eocytic lobe, eocytic gap, eocytic bulge and a modied central protuberance (Lake et al., 1984). However, it was later shown that the eocytic features were present in other archaea, such as Methanococcus vannielii (e.g. Harauz et al., 1987), and the eocytic hypothesis is no longer accepted. The small ribosomal subunit of the archaeal ribosome shares a common structural core with those of bacteria, but has additional features. The archaeal and eukaryotic small ribosomal subunits both have a feature on the head of the
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Archaeal Ribosomes

subunit called the bill (Figure 1) (Lake et al., 1984), which is situated adjacent to the stalk of the large ribosomal subunit in whole ribosomes. In bacteria, the site corresponding to the bill includes proteins (eS10, eS14 and eS3) that are implicated in transfer RNA (tRNA) recognition and binding. The function of the archaeal bill is, therefore, thought to be associated, directly or indirectly, with that of the large ribosomal subunit stalk, mainly in the elongation factor-related steps and in coordination of protein synthesis. The unique archaeal small ribosomal subunit structural features observed electron microscopically are believed to be due to a greater number of r-proteins, especially in Methanococcus and the sulfur-dependent thermophiles, than that in bacteria.

Ribosomal Proteins
Archaeal r-proteins and rRNAs serve as probes in studies of the phylogenetic relationships amongst the three distinct phenotypic subgroups of the archaea, namely the methanogens, the halophiles and the sulfur-dependent archaea. Archaeal r-proteins are smaller in size than their eukaryotic counterparts, but are larger than their equivalent bacterial r-proteins (Matheson, 1985). Structurally, a greater proportion of the archaeal r-proteins are acidic in nature, whereas the overwhelming majority of the eukaryotic and bacterial r-proteins are basic. These acidic rproteins require a high internal [K 1 ] and their number correlates with the magnitude of this concentration. They play an important role in allowing the ribosomal particle to be stable and functional under high salt conditions. The number of acidic r-proteins varies within the archaea. Generally, the extreme halophiles and certain methanogens, including Methanobacterium thermoautotrophicum and Methanobrevibacter arboriphilus, have a signicantly

higher number of acidic r-proteins than the sulfurdependent archaea (Matheson, 1985; Ramirez et al., 1993 and references therein). The structural organization of the archaeal r-protein genes resembles that found in the bacteria, i.e. clustered in operons, whereas the eukaryotic r-protein genes are dispersed throughout the genome (Ramirez et al., 1993). In addition, some archaeal r-protein operons possess open reading frames that are not present in the corresponding bacterial operons. Most of these open reading frames encode r-proteins which share a high degree of sequence similarities with eukaryotic r-proteins. Moreover, some archaeal r-protein gene operons have been reported which have no equivalents in bacteria. In general, archaeal structural r-proteins, when compared to their equivalent eukaryotic and bacterial rproteins, have more substantial sequence similarity with the eukaryotic rather than bacterial r-proteins. One example is the archaeal aL12, which is a structural component of the stalk protuberance in the large ribosomal subunit (Kopke and Wittmann-Liebold, 1989). The sequence similarity of the archaeal and the eukaryotic structural r-proteins is highest in the sulfurdependent archaea and lowest in the extreme thermophiles, whereas the reverse trend is true for the bacterial relatedness of the archaeal structural r-proteins (Ramirez et al., 1993). In contrast, r-proteins that are associated with conserved functional domains of the rRNA tend to have a high level of sequence conservation in the three kingdoms. For example, the archaeal r-protein aL2, which is believed to be functionally part of the peptidyltransferase centre, shows a great deal of sequence similarity with both bacterial eL2 and eukaryotic uL2. Other r-proteins (aL3, aL4, aL16) located in this region, which appear to be of more structural importance, show distant relatedness amongst the three domains of life.

Eukaryote L1 protuberance Peptidyltransferase site (a) Archaea Bill' 16S 3-region (b) 16S 5-region (c) Bacteria Central protuberance Stalk

Figure 1 (a) Large, and (b) small ribosomal subunits, illustrating the peptidyltransferase centre and some distinguishing features seen by electron microscopy. (c) Small ribosomal subunits from different kingdoms have the same overall structure, but with slight perceived variations.

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Archaeal Ribosomes

Ribosomal RNA
The ribosomal RNAs play an important functional role in the ribosome, in contrast to earlier beliefs that they were structural scaolds only. In archaea, each 70S ribosome contains one molecule of 5S, 16S and 23S rRNA, and many of these have been sequenced (Matheson, 1985 and references therein). There is no evidence for a separate 5.8S rRNA molecule, as is found in eukaryotic ribosomes, but the archaeal 23S rRNA contains a sequence segment at its 5-end homologous to 5.8S rRNA (Ramirez et al., 1993 and references therein). With respect to order, the archaeal rRNA gene arrangement is quite similar to that of bacteria (i.e. 16S-23S-5S). However, the relative positional organization of the archaeal rRNA genes is variable. In some archaea, as in bacteria, the three rRNA genes are closely linked, whereas other archaea have unlinked rRNA genes (Ramirez et al., 1993). Furthermore, in archaea, the number of rRNA operons varies: three in Methanococcus vannielii; two in Methanobacterium thermoautotrophicum, Methanothermus fervidus and Haloarcual marismortui (Ramirez et al., 1993). The secondary structures of the two large rRNAs (23S and 16S) from bacteria, eukaryotes and archaea show that there is a conserved structural core, containing the functional domains, with attached variable extensions at specic sites. These variable extensions contain unique structural elements that can be used to determine the phylogenetic lineage of the organism. Structurally, the 16S rRNA molecule can be divided into three major domains (5-major domain, the central domain, and 3-major domain) and a 3-minor domain, while the 23S rRNA molecule consists of six domains. In the 16S rRNA, the 3major domain functions in translational elongation and termination. The central and the 5-domains carry out subunit association and translation accuracy, respectively, and the 3-minor domain is involved in translational initiation and decoding (Ramirez et al., 1993). In the 23S rRNA, domain II comprises the guanosine triphosphatase (GTPase) centre, and certain regions of domains IV and V make up the major components of the peptidyltransferase centre. During protein synthesis, the peptidyltransferase centre catalyses the peptide bond formation between the aminoacyl-tRNA at the A site of the ribosome and the growing nascent protein at the P site of the ribosome. The GTPase centre is the site of interaction of the elongation factors with the ribosome, and is responsible for triggering the GTPase activity of the ribosome during the elongation step of protein synthesis (Ramirez et al., 1993). The 5S rRNA is universal to all life forms and shows highly conserved primary and secondary structures. The archaeal 5S rRNA shares common secondary structural features with both bacterial and eukaryotic 5S rRNA, namely the molecular stalk (I), the tuned helix (II) and the common arm base (III). Like the eukaryotic 5S rRNAs,

archaeal 5S rRNAs have dierent helical arrangements in the region corresponding to the prokaryotic loop (IV), which is a feature unique to the bacterial rRNA molecules. However, in some archaeal strains, the looped out base in helix IV is followed by two base pairs instead of the characteristic three in eukaryotes (Fox, 1985). In addition, archaeal 5S rRNA shows a putative helix V region that is structurally dierent from the eukaryotic helix V. Unlike the eukaryotic helix V, in most archaeal 5S rRNAs the putative helix V indicates no coaxial relation between either helices I and V or helices II and V.

Protein Synthesis and Targeting

In archaea, protein synthesis follows a general mechanism which shows eukaryotic- and bacterial-like features. The 16S rRNA of some archaea has a bacterial-like Shine Dalgarno (SD) sequence (Amils et al., 1993 and references therein). Frequently, the interaction between the messenger RNA (mRNA) and the archaeal ribosome is mediated by the recognition between the mRNA and the putative SD sequence. However, this SD sequence is not an obligatory feature in the halophiles and the sulfur-dependent archaea. AUG often serves as the start codon for most archaeal protein synthesis. Like eukaryotes, methionyl-tRNA is the initiator aminoacyl moiety in archaea, and the binding of incoming aminoacyl-tRNA to the ribosome is catalysed by the EF-1a elongation factor. The translocation of the peptidyl-tRNA from the ribosomal A site at the P site is catalysed by the EF-2 elongation factor. In eukaryotic cells, the targeting mechanism of secretory and membrane proteins is mediated by a signal recognition particle. This ribonucleoprotein particle consists of six polypeptides and a 7S rRNA molecule (Amils et al., 1993), and recognizes the N-terminal sequence (also known as the signal sequence) of the nascent protein as it emerges from the ribosome during translation. Binding of the SRP to the signal sequence halts the proteins synthesis until the SRP nascent proteinribosome complex reaches and interacts with a specic receptor on the cytosolic side of the rough endoplasmic reticulum. The interaction between this docking protein and the SRPnascent proteinribosome complex results in the release of the SRP and the resumption of the translational process. There is a growing body of evidence that suggests the existence of a similar targeting mechanism of the secretory and membrane proteins in archaea. Archaeal membrane and secretory proteins share a common motif at their Nterminus, which is similar to the signal sequence of the eukaryotic and bacterial secretory and membrane proteins (Gropp et al., 1992; Amils et al., 1993). In addition, archaea possess a 7S RNA molecule which has high sequence similarity with the eukaryotic 7S RNA. The sequences of the archaeal 7S and the bacterial 4.5S RNA molecules,
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Archaeal Ribosomes

namely the E. coli 4.5S RNA which has been determined to be a component of an RNP, are also related. Importantly, the archaeal 7S RNA molecule has been found to be associated with ribosomes during translation of membrane proteins. In studying the sedimentation behaviour of the halobacterial 7S RNA (from H. halobium ) and the mRNA encoding the membrane protein bacteriorhodopsin, it has been found that the two RNAs cosediment with membrane-bound polysomes during the expression of bacteriorhodopsin (Gropp et al., 1992).

properties, of which many are unique, also include features that are common to bacterial and eukaryotic ribosomes. In addition, it appears from archaeal r-protein and rRNA analyses that the archaea have diverged, as a group, more than eukaryotes and bacteria; however, the interlineage structural conservation of archaeal ribosomes supports the concept that these organisms are indeed part of a third evolutionary line of descent.

References
Amils R, Cammarano P and Londei P (1993) Translation in archaea. In: Kates M, Kushner DJ and Matheson AT (eds) The Biochemistry of Archaea, pp. 393438. Amsterdam: Elsevier. Fox GE (1985) The structure and evolution of archaebacterial ribosomal RNA. In: Woese CR and Wolfe RS (eds) The Bacteria, vol. 8, pp. 257 310. New York: Academic Press. Gropp R, Gropp F and Betlach MC (1992) Association of the halobacterial 7S RNA to the polysome correlates with expression of the membrane protein bacteriorhodopsin. Proceedings of the National Academy of Sciences of the USA, 89: 12041208. Harauz G, Stoeer-Meilicke M and van Heel M (1987) Characteristic views of prokaryotic 50S ribosomal subunits. Journal of Molecular Evolution 26: 347357. Henderson E, Oakes M, Clark MW et al. (1984) A new ribosome structure. Science 225: 510512. Kopke AKI and Wittmann-Liebold B (1989) Comparative studies of ribosomal proteins and their genes from M. vannielii and other organisms. Canadian Journal of Microbiology 35: 1120. Lake JA, Henderson E, Oakes M and Clark MW (1984) Eocytes: a new ribosome structure indicates kingdom with a close relationship to eucaryotes. Proceedings of the National Academy of Sciences of the USA 81: 37863790. Matheson AT (1985) Ribosomes of archaebacteria. In: Woese CR and Wolfe RS (eds) The Bacteria, vol. 8, pp. 345377. New York: Academic Press. Ramirez C, Kopke AKE, Yang D-C, Boeckh T and Matheson AT (1993) In: Kates M, Kushner DJ and Matheson AT (eds) The Biochemistry of Archaea, pp. 439465. Amsterdam: Elsevier. Woese CR, Magrum LJ and Fox GE (1978) Archaebacteria. Journal of Molecular Evolution 11: 245251.

Thermal and High Salt Stability

Many archaea inhabit extremely harsh environments (temperature around the boiling point of water, saturating salt concentrations, etc.). To sustain life under such dicult conditions, their cellular components must possess atypical features to be functional. The unique features which allow the archaeal ribosomes to achieve an ecient and accurate translational engine in such environments have been studied in detail for extremely thermophilic (Sulfolobus solfataricus) and halophilic (H. cutirubrum) archaea. In general, the extreme thermophiles have a considerably higher ribosomal subunit melting temperature than mesophilic ribosomal subunits of either bacteria or eukaryotes, e.g. 208C higher for S. solfataricus than for E. coli (Amils et al., 1993). Structurally, the thermophilic archaeal ribosomes have enhanced hydrophobicity of the internal core, mediated by more extensive rRNArprotein and r-proteinr-protein interactions than exist in mesophilic ones. Consequently, the thermophilic ribosomes assume a conformationally rigid and compact quaternary packing which allows the complexes eectively to resist better thermal-induced unfolding at high temperatures. This special conformation allows the organism to sustain an operational protein synthesis engine, and therefore live, at such extreme temperatures. Halobacterial ribosomes exist as monomers at salt concentrations ([K 1 ] and [Mg2 1 ] levels of 3.5 mol L 2 1 and 100 mmol L 2 1, respectively) that disrupt ribosomes of all other organisms (Amils et al., 1993). Indeed, they require such high salt conditions to retain structural integrity and function. As described previously, the halophilic ribosomes contain more acidic r-proteins than the nonhalophilic ones. It is believed that halotolerance stems from these highly acidic r-proteins, which allow the ribosome to maintain an appropriate hydration volume under such extreme salt concentrations.

Further Reading
Danson MJ, Hough DW and Lunt GG (eds) (1992) The archaebacteria: biochemistry and biotechnology. Biochemical Society Symposia 58. Hill WE, Dahlberg A, Garrett RA et al. (eds) (1990) The Ribosomes: Structure, Function and Evolution. Washington, DC: American Society for Microbiology. Kandler O and Zillig W (eds) (1986) Archaebacteria85. New York: Fischer. Kates M, Kushner DJ and Matheson AT (eds) (1993) The Biochemistry of Archaea. Amsterdam: Elsevier. Moore PB (1998) The three-dimensional structure of the ribosome and its components. Annual Review of Biophysics and Biomolecular Structure 27: 3558. Woese CR and Wolfe RS (eds) (1985) The Bacteria, vol. 8. New York: Academic Press. Yonath A and Franceschi F (1998) Functional universality and evolutionary design: insights from the structure of the ribosome. Structure 6: 679684.

Conclusion
In summary, it is evident, based on studies of the structural components of the archaeal ribosomes, that they are distinct from those found in eukaryotes and bacteria. Their

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