Kinetic Parameters for tmRNA Binding to Alanyl-tRNA Synthetase and Elongation Factor Tu from Escherichia coli †

Leiden University, Leyden, South Holland, Netherlands
Biochemistry (Impact Factor: 3.01). 04/2000; 39(10):2652-8. DOI: 10.1021/bi992439d
Source: PubMed

ABSTRACT Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins. Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value. The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower. These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.

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    • "This implies that rates of initial binding and EF-Tu activation are independent of mRNA length and that GTP is hydrolyzed when tmRNA is delivered to the ribosome, whether or not tmRNA reacts with the nascent peptide and ends up tagging it for destruction. Although this spurious GTP hydrolysis may waste energy, the cost is probably minimal given the fairly low concentration of tmRNA (Moore and Sauer, 2005) and its weak affinity for EF-Tu (Barends et al., 2000, 2001). Taken together, the kinetic studies of tmRNA show that selectivity for truncated mRNA takes place during the accommodation step, when tmRNA and SmpB dissociate from the ribosome if the C-terminal tail cannot bind into the mRNA channel. "
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    ABSTRACT: In bacteria, transfer-messenger RNA (tmRNA) and SmpB comprise the most common and effective system for rescuing stalled ribosomes. Ribosomes stall on mRNA transcripts lacking stop codons and are rescued as the defective mRNA is swapped for the tmRNA template in a process known as trans-translation. The tmRNA-SmpB complex is recruited to the ribosome independent of a codon-anticodon interaction. Given that the ribosome uses robust discriminatory mechanisms to select against non-cognate tRNAs during canonical decoding, it has been hard to explain how this can happen. Recent structural and biochemical studies show that SmpB licenses tmRNA entry through its interactions with the decoding center and mRNA channel. In particular, the C-terminal tail of SmpB promotes both EFTu activation and accommodation of tmRNA, the former through interactions with 16S rRNA nucleotide G530 and the latter through interactions with the mRNA channel downstream of the A site. Here we present a detailed model of the earliest steps in trans-translation, and in light of these mechanistic considerations, revisit the question of how tmRNA preferentially reacts with stalled, non-translating ribosomes.
    Frontiers in Microbiology 09/2014; 5:462. DOI:10.3389/fmicb.2014.00462 · 3.94 Impact Factor
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    • "Like that of tRNA, the 3′ end of tmRNA can be aminoacylated with an amino acid (alanine) by an aminoacyl-tRNA synthestase (alanyl-tRNA synthetase; Komine et al., 1994; Ushida et al., 1994). Other tRNA-like functions, such as 5′ processing by RNase P (Komine et al., 1994), binding to EF-Tu (Rudinger-Thirion et al., 1999; Barends et al., 2000, 2001; Hanawa-Suetsugu et al., 2001) and interaction with 70S ribosome (Ushida et al., 1994; Komine et al., 1996; Tadaki et al., 1996), have also been revealed. Although it is about fivefold larger than tRNA, tmRNA has no apparent anticodon, making it difficult to clarify whether and how tmRNA is involved in translation. "
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    ABSTRACT: Transfer messenger RNA (tmRNA; also known as 10Sa RNA or SsrA RNA) is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5' end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon-anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA(+) proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of non-functional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.
    Frontiers in Genetics 04/2014; 5:66. DOI:10.3389/fgene.2014.00066
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    • "However, both features are equipped with a single small RNA molecule called tmRNA (also known as 10Sa RNA or SsrA). The presence of the tRNA-like secondary structure with several tRNA-specific sequences including the 3′-terminal CCA sequence in tmRNA was first found in 1994 (Komine et al., 1994; Ushida et al., 1994; Figure 1A), and thereafter several other structural and functional similarities to tRNA, such as the aminoacylation capacity with alanine (Komine et al., 1994; Ushida et al., 1994), the binding capacity to the ribosome (Ushida et al., 1994; Tadaki et al., 1996), the 5′ processing by RNase P (Komine et al., 1994) and the binding capacity to EF-Tu after aminoacylation (Rudinger-Thirion et al., 1999; Barends et al., 2000, 2001; Hanawa-Suetsugu et al., 2001) and the presence of tRNA-specific base modifications (Felden et al., 1998), have been reported, although the anticodon has never been found in tmRNA, a few hundred nucleotides in length. The function as mRNA has been suggested by an observation that a peptide of 10-amino acid sequence encoded by tmRNA is attached to the truncated C-termini of polypeptides that are exogenously expressed in Escherichia coli with an alanine residue of unknown origin in between them (Tu et al., 1995; Keiler et al., 1996; Figure 1B). "
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    ABSTRACT: tmRNA is a bacterial small RNA having a structure resembling the upper half of tRNA and its 3' end accepts alanine followed by binding to EF-Tu like tRNA. Instead of lacking a lower half of the cloverleaf structure including the anticodon, tmRNA has a short coding sequence for tag-peptide that serves as a target of cellular proteases. An elaborate coordination of two functions as tRNA and mRNA facilitates an irregular translation termed trans-translation: a single polypeptide is synthesized from two mRNA molecules. It allows resumption of translation stalled on a truncated mRNA, producing a chimeric polypeptide comprising the C-terminally truncated polypeptide derived from truncated mRNA and the C-terminal tag-peptide encoded by tmRNA. Trans-translation promotes recycling of the stalled ribosomes in the cell, and the resulting C-terminally tagged polypeptide is preferentially degraded by cellular proteases. Biochemical studies using in vitro trans-translation systems together with structural studies have unveiled the molecular mechanism of trans-translation, during which the upper and lower halves of tRNA are mimicked by the tRNA-like structure of tmRNA and a tmRNA-specific binding protein called SmpB, respectively. They mimic not only the tRNA structure but also its behavior perhaps at every step of the trans-translation process in the ribosome. Furthermore, the C-terminal tail of SmpB, which is unstructured in solution, occupies the mRNA path in the ribosome to play a crucial role in trans-translation, addressing how tmRNA·SmpB recognizes the ribosome stalled on a truncated mRNA.
    Frontiers in Microbiology 02/2014; 5:65. DOI:10.3389/fmicb.2014.00065 · 3.94 Impact Factor
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