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A Mechanism for Stop Codon Recognition by the Ribosome: a Bioinformatic Approach

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A Mechanism for Stop Codon Recognition by the Ribosome: a Bioinformatic Approach Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press RNA (2001), 7:1683–1692+ Cambridge University Press+ Printed in the USA+ Copyright © 2001 RNA Society+ DOI: 10+1017+S1355838201010093 HYPOTHESIS A mechanism for stop codon recognition by the ribosome: A bioinformatic approach VALERY IVANOV,1,2 ARTEMY BENIAMINOV,1 ANDREI MIKHEYEV,2 and ELVIRA MINYAT1 1 V+A+ Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia 2 Moscow Institute of Physics and Technology, 141700 Moscow Region, Dolgoprudny, Russia ABSTRACT Protein synthesis in ribosomes requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. Synthesis is also assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by their anticodons is known. The termination factors are thought to do this. In the large ribosomal RNA, we found two sites that, like tRNAs, contain the anticodon hairpin but with triplets complementary to stop codons. One site is hairpin 69 from domain IV; the other site is hairpin 89, domain V. By analogy, we call them termination tRNAs: Ter-tRNA1 and Ter-tRNA2, respectively, even though they transport no amino acids, and suggest that they directly pair to stop codons. The termination factors only aid in this recognition, making it specific and reliable. A strong argument in favor of our hypothesis comes from vertebrate mitochondria. They are known to acquire two new stop codons, AGA and AGG. In the standard code, these are two out of six arginine codons. We revealed that the corresponding anticodons, UCU and CCU, have evolved in Ter-tRNA1 of these mitochondria. Keywords: genetic code; large rRNA; phylogeny; RNA world; termination; Ter-tRNA; translation; vertebrate mitochondria INTRODUCTION: FOUNDATION OF THE There are two alternative opinions: (1) the RFs only HYPOTHESIS recognize stop codons, and (2) the triplets (anticodons) in the small rRNA directly interact with stop codons Termination is the final process of a ribosomal cycle (though with the help of RFs; Tate & Brown, 1992)+ The and begins with the recognition of a stop codon in the first opinion prevails and is strongly supported by the mRNA by the ribosome+ This is followed by hydrolysis finding that RF2 forms crosslinks with a piece of mRNA of an ester bond that connects the complete polypep- near the decoding site (Brown & Tate, 1994)+ Moreover, tide chain to the peptidyl tRNA, removal of the deacyl- the tripeptides in RF1 and RF2 were localized, and ated tRNA from the ribosome, and dissociation of the when mutated, affected specificity of stop codon rec- latter into small and large subunits+ Obligatory partici- ognition (Ito et al+, 2000; we shall discuss this important pants of the termination process are proteins called the article further in a special section)+ release factors (RFs)+ The eubacterial proteins RF1 The site of protein synthesis in the ribosome is formed and RF2 participate in recognition of stop codons: RF1 by several structural domains of the large rRNA+ Two of works with UAG and UAA, whereas RF2 traces UGA them, IV and V (see Fig+ 1) are discussed in this work+ and UAA (Scolnick et al+, 1968)+ A single eukaryotic We paid attention to hairpin 69 (domain IV), whose protein, eRF1, serves all the three codons: UAG, UGA, terminal loop consists of 7 nt+ It, like the anticodon loop and UAA (reviewed by Kisselev & Buckingham, 2000)+ of tRNAs, has the triplet (CUA) in the middle of the loop exactly as tRNA does+ In this case, it is for a stop codon Reprint requests to present address: Valery Ivanov, Center for , + + , , UAG rather than for an amino acid codon (Fig 1) Biophysical Sciences and Engineering University of Alabama Bir- , , mingham, CBSE 221, 1530 3rd Avenue South, Birmingham, Ala- Moreover hairpin 69 together with its close neigh- bama 35294-4400, USA; e-mail: irina@uab+edu+ bors, can be depicted as a clover leaf, typical for tRNAs 1683 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1684 V. Ivanov et al. FIGURE 1. Domains IV and V of E. coli 23S rRNA+ Small numbers are the nucleotide positions+ Small bars mark every 10th nucleotide+ Large numbers designate relevant hairpins+ Stop anticodons in 7-nt loops of hairpins 69 and 89 are shown in a magnified view near the corresponding stop codons and release factors+ Three relevant crosslinks are shown by the slanted lines (Mueller et al+, 2000)+ Curved two-headed arrows connect the segments with possible stacking+ A zigzag line indicates the crosslink between the loop of hairpin 69 and 16S rRNA (decoding site)+ Wower’s crosslink (J+ Wower, pers+ comm+) is seen above hairpin 89+ A set of rectangular lines below the hairpin 89 is WC-pairing in a suggested pseudoknot (Ivanov et al+, 1999)+ For all the details see text+ (see Fig+ 2A)+ Here, it is a mimic of a tyrosine su(13) or Ter-tRNA1+ Interestingly, m2G, which is frequently Tyr-tRNA (Fig+ 2B)+ It is clear that the number of iden- met in the 10th position in tRNAs at the bottom of a tical nucleotides, having identical positions (except for DHU-stem (Saenger, 1984), is similarly disposed in the the CCA stem) is too great to be just a random coinci- Ter-tRNA1 (Fig+ 2A)+ dence+ Especially impressive is the similarity between There is a Ter-tRNA2 as well, which is found in do- the TCC-stem and its ribosomal mimic+ We termed main V (Fig+ 1)+ In this case, it is confined to hairpin 89, these combined four rRNA hairpins the termination tRNA whose 7-nt terminal loop imitates the tRNA anticodon Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Stop codon recognition with large rRNA 1685 FIGURE 2. A clover leaf depiction for hairpins 68–71 of E. coli 23S rRNA (A) and su(13)Tyr-tRNA (B)+ Identical elements are shaded+ Below, see pairing of the stop codon with its anticodon+ loop containing the anticodon UCA for the stop codon 3+ Are stop anticodons present in hairpins 69 and 89 in UGA+ organisms of different taxons or not? We formulate the following hypothesis: ARCHIVE INFORMATION Stop codons in mRNA are recognized by the stop anticodons of the tRNA mimics, Ter-tRNA1 and The data we used on the large rRNA sequences are Ter-tRNA2, covalently built into the large rRNA, available at the sites www+rna+icmb+utexas+edu and and termination proteins RF1 and RF2 promote http://rrna+uia+ac+be/lsu+ We have analyzed more than this recognition by increasing its accuracy and 500 sequences belonging to bacteria, eukaryotes, chlo- fidelity. roplasts, and mitochondria+ A search for the particular regions including hairpins 69 and 89 and their neigh- In this article, we restrict ourselves to the consider- borhood was facilitated by the fact that they are the ation of termination in bacteria and organelles, mito- most conserved regions in the large rRNA+ chondria, and chloroplasts+ These organelles are far Atom coordinates of the Haloarcula marismortui 50S descendants of bacteria that formerly inhabited eukary- subunit (Ban et al+, 2000) were obtained from the Pro- otic cells+ Presently we do not exclude that this concept tein Data Bank, accession number 1FFK+ The program is also true for eukaryota and archaebacteria+ How- Insight 2 for the workstation Silicon Graphics displayed ever, the protein mimics of RNA in eukaryota might go the three-dimensional structures, which are necessary so far to develop entirely “proteinic” recognition of stop for answering Crucial Question 1+ codons, so that this mechanism no longer exists in higher organisms+ ANSWERING THE CRUCIAL QUESTIONS 1+ Are hairpins 69 and 89 present near mRNA in the CRUCIAL QUESTIONS A-site, at the interface between the small and large Prior to specifying the announced hypothesis and in- ribosomal subunits, or not? specting its predictions, three crucial questions must be answered: The data obtained by crosslinking summarized in Muel- ler et al+ (2000; see also Fig+ 1) help to answer this 1+ Are hairpins 69 and 89 present near mRNA in the question regarding hairpin 69 with optimism: 23S rRNA A-site, at the interface between the small and large hairpin 69 (nt 1911–1920) crosslinks to 16S rRNA hair- ribosomal subunits, or not? pin 44 (nt 1408–1411)+ In addition, according to the 2+ Can the noncanonical pair A-C be formed, when data of X-ray studies (Cate et al+, 1999; Yusupov et al+, necessary, during pairing of stop codons and stop 2001), the loop of the hairpin 69 makes direct contact anticodons or not? (Such a pair is necessary for the with the decoding site in the small subunit (hairpin 44, anticodons of hairpins 69 and 89 to pair with the nt 1494 and 1408–1410)+ Unfortunately, the X-ray data UAA stop codon+) from the large subunit (Ban et al+, 2000) does not show Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press 1686 V. Ivanov et al. the loop of the hairpin 69+ In this respect, the recent CmCA inherent to Trp-tRNA+ In a test of binding to a paper by Yusupov et al+ (2001) is especially important+ ribosome, this tRNA binds to a stop codon UGA, but It confirms cryo-electron microscopy data on the close not to serine or tryptophan codons+ In vitro experi- proximity between the loop of helix 69 and the anti- ments confirmed that this tRNA exhibits suppressor ac- codon hairpin of A-site tRNA, but also testifies to the tivity toward the UGA codon+ In this connection, one agile nature of hairpin 69+ Because the secondary struc- can be reminded that a suppressor Trp-tRNA from ture of rRNA is universal for different species of organ- E.
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