Prokaryotic large ribosomal subunit

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Atomic structure of the 50S Subunit from Haloarcula marismortui. Proteins are shown in blue and the two RNA strands in orange and yellow. The small patch of green in the center of the subunit is the active site. 010 large subunit-1FFK.gif
Atomic structure of the 50S Subunit from Haloarcula marismortui. Proteins are shown in blue and the two RNA strands in orange and yellow. The small patch of green in the center of the subunit is the active site.
Atomic structure of the 50S large subunit of the ribosome, facing the 30S small ribosomal subunit. Proteins are colored in blue and RNA in ochre. The active site, adenine 2486, is highlighted in red. Image created from PDB: 3CC2 using PyMol 50S-subunit of the ribosome 3CC2.png
Atomic structure of the 50S large subunit of the ribosome, facing the 30S small ribosomal subunit. Proteins are colored in blue and RNA in ochre. The active site, adenine 2486, is highlighted in red. Image created from PDB: 3CC2 ​ using PyMol

50S is the larger subunit of the 70S ribosome of prokaryotes, i.e. bacteria and archaea. It is the site of inhibition for antibiotics such as macrolides, chloramphenicol, clindamycin, and the pleuromutilins. It includes the 5S ribosomal RNA and 23S ribosomal RNA.

Contents

Despite having the same sedimentation rate, bacterial and archaeal ribosomes can be quite different.

Structure

50S, roughly equivalent to the 60S ribosomal subunit in eukaryotic cells, is the larger subunit of the 70S ribosome of prokaryotes. The 50S subunit is primarily composed of proteins but also contains single-stranded RNA known as ribosomal RNA (rRNA). rRNA forms secondary and tertiary structures to maintain the structure and carry out the catalytic functions of the ribosome.

X-ray crystallography has yielded electron density maps allowing the structure of the 50S in Haloarcula marismortui (archaeon) to be determined to 2.4Å resolution [1] and of the 50S in the Deinococcus radiodurans (bacterium) to 3.3Å. [2] The large ribosomal subunit (50S) is approximately twice as massive as the small ribosomal subunit (30S). The model of Hm 50S, determined in 2000 by Nenad Ban and colleagues in the laboratory of Thomas Steitz and the laboratory of Peter Moore, includes 2711 of the 2923 nucleotides of 23S rRNA, all 122 nucleotides of its 5S rRNA, and structure of 27 of its 31 proteins. [1]

Ribosomal RNA

The secondary structure of 23S is divided into six large domains, within which domain V is most important in its peptidyl transferase [3] activity. Each domain contains normal secondary structure (e.g., base triple, tetraloop, cross-strand purine stack) and is also highly symmetric in tertiary structure; proteins intervene between their helices. At tertiary structure level, the large subunit rRNA is a single gigantic domain while the small subunit contains three structural domains. This difference reflects the lesser flexibility of the large subunit required by its function. While its core is conserved, it accommodates expansion segments on its periphery. [4] [5]

Difference between bacteria and archaeal versions

A cryoEM structure of the 50S subunit from the archaeon Methanothermobacter thermautotrophicus has been determined. It shares the 50S size/sedimentation rate and the two rRNA count, but its 23S expansion segments have more in common with eukaryotes. [6]

A cryoEM reconstruction of the native 50S subunit of the extremely halophilic Archaean Halococcus morrhuae (classified under Euryarchaeota; Stenosarchaea group) is available. The 50S subunit contains a 108‐nucleotide insertion in its 5S rRNA, [7] which at subnanometer resolution, is observed to emerge from a four‐way junction without affecting the parental canonical 5S rRNA structure. [4]

Due to the differences, archaeal 50S are less sensitive to some antibiotics that target bacterial 50S. [8] [9]

Function

50S includes the activity that catalyzes peptide bond formation (peptidyl transfer reaction), prevents premature polypeptide hydrolysis, provides a binding site for the G-protein factors (assists initiation, elongation, and termination), and helps protein folding after synthesis.

Peptidyl transfer reaction

An induced-fit mechanism has been revealed for how 50S catalyzes the peptidyl transfer reaction and prevents peptidyl hydrolysis. The amino group of an aminoacyl-tRNA (binds to A site) attacks the carbon of a carbonyl group of a peptidyl-tRNA (binds to P site) and finally yields a peptide extended by one amino acid esterified to the A site tRNA bound to the ribosomal A site and a deacylated tRNA in the P site.

When the A site is unoccupied, nucleotide U2620 (E. coli U2585), A2486 (2451) and C2106 (2063) sandwich the carbonyl group in the middle, forcing it into an orientation facing the A site. This orientation prevents any nucleophilic attack from the A site because the optimal attacking angle is 105 degrees from the plane of the ester group. When a tRNA with a complete[?] CCA sequence at its acceptor stem is bound to the A site, C74 of the tRNA stacking with U2590 (2555) induces a conformational change in the ribosome, resulting in movement of U2541 (2506), U2620 (2585) through G2618 (2583). The displacement of bases allows the ester group to adopt a new conformation accessible to nucleophilic attack from the A site.

The N3 (nitrogen) of A2486 (2451) is closest to the peptide bond being synthesized and may function as a general base to facilitate the nucleophilic attack by the amino group of the aminoacyl-tRNA (in the A site). The pKa of A2486 (2451) is about 5 units higher in order to hydrogen bond with the amino group thus increasing its nucleophilicity. The elevation of pKa is achieved through a charge relay mechanism. A2486 (2451) interacts with G2482 (G2447), which hydrogen bonds with the buried phosphate of A2486 (2450). This buried phosphate can stabilize the normally rare imino tautomers of both bases, resulting in an increase in the negative charge density on N3.

Protein assembly

After initiation, elongation, and termination, there is a fourth step of the disassembly of the post-termination complex of ribosome, mRNA, and tRNA, which is a prerequisite for the next round of protein synthesis. The large ribosomal subunit has a role in protein folding both in vitro and in vivo . The large ribosomal subunit provides a hydrophobic surface for the hydrophobic collapse step of protein folding. The newly synthesized protein needs full access to the large subunit to fold; this process may take a period of time (5 minutes for beta-galactosidase [ citation needed ]).

See also

Related Research Articles

<span class="mw-page-title-main">RNA</span> Family of large biological molecules

Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself or by forming a template for the production of proteins. RNA and deoxyribonucleic acid (DNA) are nucleic acids. The nucleic acids constitute one of the four major macromolecules essential for all known forms of life. RNA is assembled as a chain of nucleotides. Cellular organisms use messenger RNA (mRNA) to convey genetic information that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.

<span class="mw-page-title-main">Ribosome</span> Synthesizes proteins in cells

Ribosomes are macromolecular machines, found within all cells, that perform biological protein synthesis. Ribosomes link amino acids together in the order specified by the codons of messenger RNA molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA molecules and many ribosomal proteins. The ribosomes and associated molecules are also known as the translational apparatus.

<span class="mw-page-title-main">Translation (biology)</span> Cellular process of protein synthesis

In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in the addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.

<span class="mw-page-title-main">Ribosomal RNA</span> RNA component of the ribosome, essential for protein synthesis in all living organisms

Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal DNA (rDNA) and then bound to ribosomal proteins to form small and large ribosome subunits. rRNA is the physical and mechanical factor of the ribosome that forces transfer RNA (tRNA) and messenger RNA (mRNA) to process and translate the latter into proteins. Ribosomal RNA is the predominant form of RNA found in most cells; it makes up about 80% of cellular RNA despite never being translated into proteins itself. Ribosomes are composed of approximately 60% rRNA and 40% ribosomal proteins, though this ratio differs between prokaryotes and eukaryotes.

The peptidyl transferase center is an aminoacyltransferase ribozyme located in the large subunit of the ribosome. It forms peptide bonds between adjacent amino acids during the translation process of protein biosynthesis. It is also responsible for peptidyl-tRNA hydrolysis, allowing the release of the synthesized peptide chain at the end of translation. Peptidyl transferase activity is not mediated by any ribosomal proteins, but entirely by ribosomal RNA (rRNA). The peptidyl transferase center is a significant piece of evidence supporting the RNA World hypothesis.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

<span class="mw-page-title-main">Ribosomal protein</span> Proteins found in ribosomes

A ribosomal protein is any of the proteins that, in conjunction with rRNA, make up the ribosomal subunits involved in the cellular process of translation. E. coli, other bacteria and Archaea have a 30S small subunit and a 50S large subunit, whereas humans and yeasts have a 40S small subunit and a 60S large subunit. Equivalent subunits are frequently numbered differently between bacteria, Archaea, yeasts and humans.

<span class="mw-page-title-main">5S ribosomal RNA</span> RNA component of the large subunit of the ribosome

The 5S ribosomal RNA is an approximately 120 nucleotide-long ribosomal RNA molecule with a mass of 40 kDa. It is a structural and functional component of the large subunit of the ribosome in all domains of life, with the exception of mitochondrial ribosomes of fungi and animals. The designation 5S refers to the molecule's sedimentation coefficient in an ultracentrifuge, which is measured in Svedberg units (S).

Ribosomal particles are denoted according to their sedimentation coefficients in Svedberg units. The 60S subunit is the large subunit of eukaryotic 80S ribosomes, with the other major component being the eukaryotic small ribosomal subunit (40S). It is structurally and functionally related to the 50S subunit of 70S prokaryotic ribosomes. However, the 60S subunit is much larger than the prokaryotic 50S subunit and contains many additional protein segments, as well as ribosomal RNA expansion segments.

<span class="mw-page-title-main">23S ribosomal RNA</span> A component of the large subunit of the prokaryotic ribosome

The 23S rRNA is a 2,904 nucleotide long component of the large subunit (50S) of the bacterial/archean ribosome and makes up the peptidyl transferase center (PTC). The 23S rRNA is divided into six secondary structural domains titled I-VI, with the corresponding 5S rRNA being considered domain VII. The ribosomal peptidyl transferase activity resides in domain V of this rRNA, which is also the most common binding site for antibiotics that inhibit translation, making it a target for ribosomal engineering. A well-known member of this antibiotic class, chloramphenicol, acts by inhibiting peptide bond formation, with recent 3D-structural studies showing two different binding sites depending on the species of ribosome. Numerous mutations in domains of the 23S rRNA with Peptidyl transferase activity have resulted in antibiotic resistance. 23S rRNA genes typically have higher sequence variations, including insertions and/or deletions, compared to other rRNAs.

<span class="mw-page-title-main">EF-G</span> Prokaryotic elongation factor

EF-G is a prokaryotic elongation factor involved in mRNA translation. As a GTPase, EF-G catalyzes the movement (translocation) of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome.

The eukaryotic small ribosomal subunit (40S) is the smaller subunit of the eukaryotic 80S ribosomes, with the other major component being the large ribosomal subunit (60S). The "40S" and "60S" names originate from the convention that ribosomal particles are denoted according to their sedimentation coefficients in Svedberg units. It is structurally and functionally related to the 30S subunit of 70S prokaryotic ribosomes. However, the 40S subunit is much larger than the prokaryotic 30S subunit and contains many additional protein segments, as well as rRNA expansion segments.

<span class="mw-page-title-main">Protein synthesis inhibitor</span> Inhibitors of translation

A protein synthesis inhibitor is a compound that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins.

<span class="mw-page-title-main">Eukaryotic ribosome</span> Large and complex molecular machine

Ribosomes are a large and complex molecular machine that catalyzes the synthesis of proteins, referred to as translation. The ribosome selects aminoacylated transfer RNAs (tRNAs) based on the sequence of a protein-encoding messenger RNA (mRNA) and covalently links the amino acids into a polypeptide chain. Ribosomes from all organisms share a highly conserved catalytic center. However, the ribosomes of eukaryotes are much larger than prokaryotic ribosomes and subject to more complex regulation and biogenesis pathways. Eukaryotic ribosomes are also known as 80S ribosomes, referring to their sedimentation coefficients in Svedberg units, because they sediment faster than the prokaryotic (70S) ribosomes. Eukaryotic ribosomes have two unequal subunits, designated small subunit (40S) and large subunit (60S) according to their sedimentation coefficients. Both subunits contain dozens of ribosomal proteins arranged on a scaffold composed of ribosomal RNA (rRNA). The small subunit monitors the complementarity between tRNA anticodon and mRNA, while the large subunit catalyzes peptide bond formation.

The P-site is the second binding site for tRNA in the ribosome. The other two sites are the A-site (aminoacyl), which is the first binding site in the ribosome, and the E-site (exit), the third. During protein translation, the P-site holds the tRNA which is linked to the growing polypeptide chain. When a stop codon is reached, the peptidyl-tRNA bond of the tRNA located in the P-site is cleaved releasing the newly synthesized protein. During the translocation step of the elongation phase, the mRNA is advanced by one codon, coupled to movement of the tRNAs from the ribosomal A to P and P to E sites, catalyzed by elongation factor EF-G.

<span class="mw-page-title-main">Nucleic acid quaternary structure</span>

Nucleic acidquaternary structure refers to the interactions between separate nucleic acid molecules, or between nucleic acid molecules and proteins. The concept is analogous to protein quaternary structure, but as the analogy is not perfect, the term is used to refer to a number of different concepts in nucleic acids and is less commonly encountered. Similarly other biomolecules such as proteins, nucleic acids have four levels of structural arrangement: primary, secondary, tertiary, and quaternary structure. Primary structure is the linear sequence of nucleotides, secondary structure involves small local folding motifs, and tertiary structure is the 3D folded shape of nucleic acid molecule. In general, quaternary structure refers to 3D interactions between multiple subunits. In the case of nucleic acids, quaternary structure refers to interactions between multiple nucleic acid molecules or between nucleic acids and proteins. Nucleic acid quaternary structure is important for understanding DNA, RNA, and gene expression because quaternary structure can impact function. For example, when DNA is packed into heterochromatin, therefore exhibiting a type of quaternary structure, gene transcription will be inhibited.

<span class="mw-page-title-main">Elongation factor P</span> Protein domain

EF-P is an essential protein that in bacteria stimulates the formation of the first peptide bonds in protein synthesis. Studies show that EF-P prevents ribosomes from stalling during the synthesis of proteins containing consecutive prolines. EF-P binds to a site located between the binding site for the peptidyl tRNA and the exiting tRNA. It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. The EF-P protein shape and size is very similar to a tRNA and interacts with the ribosome via the exit “E” site on the 30S subunit and the peptidyl-transferase center (PTC) of the 50S subunit. EF-P is a translation aspect of an unknown function, therefore It probably functions indirectly by altering the affinity of the ribosome for aminoacyl-tRNA, thus increasing their reactivity as acceptors for peptidyl transferase.

<span class="mw-page-title-main">Helix 69</span>

Helix 69 is a hairpin RNA structure containing 19 nucleotides in large subunit of the ribosome. Ribosome consists of large and small subunits joined with inter subunit bridges. Helix 69 interacts with the helix 44 (h44) of the small subunit to form the largest interface of two subunits called inter-subunit bridge B2a, one of the most conserved regions of the ribosome. Helix 69 is proposed to be a good drug target for antibacterial drugs. Many of the recent crystal structures have shown the involvement of this hairpin in different stages of the protein translation process. By targeting bacterial helix 69 specifically, protein synthesis in bacteria could be halted thus killing the bacteria.

Ribosomal L28e protein family is a family of evolutionarily related proteins. Members include 60S ribosomal protein L28.

<span class="mw-page-title-main">Nenad Ban</span> Croatian biochemist

Nenad Ban is a biochemist born in Zagreb, Croatia who currently works at the ETH Zurich, Swiss Federal Institute of Technology, as a professor of Structural Molecular Biology. He is a pioneer in studying gene expression mechanisms and the participating protein synthesis machinery.

References

  1. 1 2 3 Nissen, P.; Hansen, J.; Ban, N.; Moore, P.; Steitz, T. (2000). "The complete atomic structure of the large ribosomal subunit at 2.4 A resolution". Science. 289 (5481): 905–920. Bibcode:2000Sci...289..905B. CiteSeerX   10.1.1.58.2271 . doi:10.1126/science.289.5481.905. PMID   10937989.
  2. ↑ Schluenzen, F.; Tocilj, A.; Zarivach, R.; Harms, J.; Gluehmann, M.; Janell, D.; Bashan, A.; Bartels, H.; Agmon, I.; Franceschi, F.; Yonath, A. (2000). "Structure of functionally activated small ribosomal subunit at 3.3 Å resolution". Cell. 102 (5): 615–623. doi: 10.1016/S0092-8674(00)00084-2 . PMID   11007480.
  3. ↑ Tirumalai MR, Rivas M, Tran Q, Fox GE (November 10, 2021). "The Peptidyl Transferase Center: a Window to the Past". Microbiol Mol Biol Rev. 85 (4): e0010421. doi:10.1128/MMBR.00104-21. PMC   8579967 . PMID   34756086.
  4. 1 2 Tirumalai, MR; Kaelber, JT; Park, DR; Tran, Q; Fox, GE (31 August 2020). "Cryo-electron microscopy visualization of a large insertion in the 5S ribosomal RNA of the extremely halophilic archaeon Halococcus morrhuae". FEBS Open Bio. 10 (10): 1938–1946. doi: 10.1002/2211-5463.12962 . PMC   7530397 . PMID   32865340.
  5. ↑ Penev PI, Fakhretaha-Aval S, Patel VJ, Cannone JJ, Gutell RR, Petrov AS, Williams LD, Glass JB (August 2020). "Supersized ribosomal RNA expansion segments in Asgard archaea". Genome Biology and Evolution. 12 (10): 1694–1710. doi: 10.1093/gbe/evaa170 . PMC   7594248 . PMID   32785681.
  6. ↑ Greber, Basil J.; Boehringer, Daniel; Godinic-Mikulcic, Vlatka; Crnkovic, Ana; Ibba, Michael; Weygand-Durasevic, Ivana; Ban, Nenad (May 2012). "Cryo-EM Structure of the Archaeal 50S Ribosomal Subunit in Complex with Initiation Factor 6 and Implications for Ribosome Evolution". Journal of Molecular Biology. 418 (3–4): 145–160. doi: 10.1016/j.jmb.2012.01.018 . PMC   3879142 . PMID   22306461.
  7. ↑ Luehrsen, KR.; Nicholson, DE; Eubanks, DC; Fox, GE (May 1981). "An archaebacterial 5S rRNA contains a long insertion sequence". Nature. 293 (5835): 755–756. Bibcode:1981Natur.293..755L. doi:10.1038/293755a0. PMID   6169998. S2CID   4341755.
  8. ↑ Khelaifia, S.; Drancourt, M. (September 2012). "Susceptibility of archaea to antimicrobial agents: applications to clinical microbiology". Clinical Microbiology and Infection. 18 (9): 841–848. doi: 10.1111/j.1469-0691.2012.03913.x . PMID   22748132.
  9. ↑ Thombre, Rebecca S.; Shinde, Vinaya; Thaiparambil, Elvina; Zende, Samruddhi; Mehta, Sourabh (13 September 2016). "Antimicrobial Activity and Mechanism of Inhibition of Silver Nanoparticles against Extreme Halophilic Archaea". Frontiers in Microbiology. 7: 1424. doi: 10.3389/fmicb.2016.01424 . PMC   5020055 . PMID   27679615. The haloarchaea used in the current study were resistant to nalidixic acid, streptomycin, gentamicin, tetracycline, erythromycin, chloramphenicol, cephalothin, and clindamycin.
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