Release factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis

doi:10.1016/S0167-4781(00)00162-7

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Volume 1493, Issues 1–2, 7 September 2000, Pages 1-11

https://doi.org/10.1016/S0167-4781(00)00162-7 Get rights and content

Abstract

The decoding of stop signals in mRNA requires protein release factors. Two classes of factor are found in both prokaryotes and eukaryotes, a decoding factor and a stimulatory recycling factor. These factors form complexes at the active centre of the ribosome and mimic in overall shape the complexes found at other stages of protein synthesis. The decoding release factor is shaped like a tRNA and has a domain for codon recognition at the decoding site of the ribosome, and a domain for peptidyl-tRNA hydrolysis that is inferred to be near the peptidyltransferase centre. Initial interaction of the decoding factor with the ribosome is a low fidelity event involving multiple contacts with the ribosomal components. A subsequent discrimination step, at present poorly defined, ensures high fidelity of codon recognition.

Introduction

Decoding of signals in mRNA takes place on the ribosome and, collectively, many resident and visiting proteins and RNA molecules are directly or indirectly important to the process. For many years now, detailed studies on individual components have accumulated a wealth of knowledge that is like pieces of a jigsaw in understanding the overall decoding processes. There have been two ‘key’ breakthroughs that helped to put the jigsaw together. The first was largely conceptual and was the gradual recognition that the RNAs of the ribosome, rather than the many proteins, were the fundamental molecules critical for function and that the protoribosome was probably simply an RNA molecule [1]. This followed from the idea of an ‘RNA World’, a concept stimulated by the discovery that RNA could catalyse reactions as well as store information [2], [3]. From that time in the mid 1980s, there has been a significant focus on rRNAs and their importance in translation. The fact that the code was contained in an RNA molecule and that the main decoding molecules of translation were tRNAs functioning in initiation and elongation, strongly reinforced this concept. However, the termination mechanism remained an enigma because protein release factors (RFs) rather than RNAs seemed to be the direct participants in decoding and no tRNAs specific for termination have been found. There was the strong possibility that a specific decoding mechanism for this step of protein synthesis was a late ‘add-on’ and previously an unassigned or genuine nonsense codon signalled the complex to ‘fall-off’ the ribosome as a default termination mechanism. We now view this process somewhat differently as a result of recent advances in the understanding of ribosomal structure.

Section snippets

Structural mimicry among translational complexes

Two technological ‘tours de force’ have provided the second ‘key’ breakthrough. The advent of cryoelectron microscopy giving high resolution structures from the latter part of the 1990’s [4], [5], [6] and the very recent X-ray crystallography solutions of the small ribosomal subunit [7], [8], large ribosomal subunit [9] and the whole ribosome [10], have brought us much closer to understanding protein synthesis in atomic detail. Firstly, with both techniques, individual helices of the RNA and

Functional domains of the class I RFs

If the decoding RFs are true ‘mimics’ of a tRNA, they must interact with the decoding site of the small ribosomal subunit and the peptidyltransferase centre of the large ribosomal subunit. In addition, it is likely that they make other contacts with the ribosomal active centre. The dimension of the eRF1 structure is such that it could span the decoding site and the peptidyltransferase centre like a tRNA. Domain 1 is inferred to interact with the stop signal in the decoding site and domain 2

Fidelity of polypeptide release

The RF can bind to the ribosome as a result of many interactions between parts of its structure and rRNA nucleotides and residues of the ribosomal proteins. To date, there has been no detailed analysis of the points of close contact with rRNA, although a RF footprint has been found to cover nucleotides of the α-sarcin loop of the large subunit rRNA (C.M. Brown and W.P. Tate, unpublished data). In addition, some nucleotides in rRNA have been shown to influence termination [39]. On the other

Possible mechanisms for translation termination fidelity

In reality, in the bacterial cell, the EF ternary complexes are of sufficient concentration to compete effectively and exclude RFs from the ribosomal active centre at non-cognate or near-cognate codons. However, this alone is unlikely to be sufficient to account for the high degree of fidelity measured in vivo [49]. Also, it cannot explain the discrimination between the stop signals themselves by the two bacterial factors, RF1 and RF2. There is more than one feasible explanation that could

Acknowledgments

The work of our own laboratory described here has been supported by the Marsden Fund of New Zealand, the Health Research Council of New Zealand, the Human Frontier Science Program (Grants RG 416/93 awarded to Y. Nakamura and WPT and RG32/97 awarded to Y. Nakamura, L. Kisselev, M. Philippe and W.P.T.) and a Howard Hughes International Investigator award to W.P.T.

References (52)

K. Kruger et al.
Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena
Cell
(1982)
C. Guerrier-Takada et al.
The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme
Cell
(1983)
H. Stark et al.
Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy
Cell
(1997)
I.S. Gabashvili et al.
Solution structure of the E. coli 70S ribosome at 11.5 Å resolution
Cell
(2000)
C.M. Brown et al.
Direct recognition of mRNA stop signals by Escherichia coli polypeptide chain release factor 2
J. Biol. Chem.
(1994)
J.G. Moffat et al.
A single proteolytic cleavage in release factor 2 stabilizes ribosome binding and abolishes peptidyl-tRNA hydrolysis activity
J. Biol. Chem.
(1994)
G. Grentzmann et al.
Function of polypeptide chain release factor 3 in Escherichia coli
J. Biol. Chem.
(1995)
D.S. Konecki et al.
Characterization of reticulocyte release factor
J. Biol. Chem.
(1977)
H. Song et al.
The crystal structure of human eukaryotic release factor eRF1-Mechanism of stop codon recognition and peptidyl-tRNA hydrolysis
Cell
(2000)
W.P. Tate et al.
The Escherichia coli ribosomal protein L11 supresses release factor 2 but promotes release factor 1 activities in peptide chain termination
J. Biol. Chem.
(1983)

W.P. Tate et al.

The NH₂-terminal domain of Escherichia coli ribosomal protein L11

J. Biol. Chem.

(1984)

D. Moazed et al.

Specific protection of 16S rRNA by translational initiation factors

J. Mol. Biol.

(1995)

W.P. Tate et al.

The translational stop signal: codon with a context or extended factor recognition element?

Biochimie

(1996)

I.L. Armstrong et al.

The requirement for the Escherichia coli ribosomal proteins L7 and L12 in release factor-dependent peptide chain termination

FEBS Lett.

(1980)

S. Schilling-Bartetzko et al.

Apparent association constants of tRNAs for the ribosomal A, P, and E sites

J. Biol. Chem.

(1992)

B. Kastner et al.

Localization of the release factor-2 binding site on 70S ribosomes by immuno-electron microscopy

J. Mol. Biol.

(1990)

M.E. Askarian-Amiri et al.

Functional characterization of yeast mitochondrial release factor 1

J. Biol. Chem.

(2000)

M.Yu. Pavlov et al.

Rate of translation of natural mRNAs in an optimised in vitro system

Arch. Biochem. Biophys.

(1996)

F. Jørgensen et al.

Release factor-dependent false stops are infrequent in Escherichia coli

J. Mol. Biol.

(1993)

G. Grentzmann et al.

Ribosomal binding site of release factors RF1 and RF2

J. Biol. Chem.

(1997)

P.B. Moore

The ribosome returns

Nature

(1988)

R.K. Agrawal et al.

Vizualisation of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation

Proc. Natl. Acad. Sci. USA

(1998)

W.M. Clemons et al.

Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution

Nature

(1999)

A. Tocilj et al.

The small ribosomal subunit from Thermus thermophilus at 4.5 Å resolution: pattern fittings and the identification of a functional site

Proc. Natl. Acad. Sci. USA

(1999)

N. Ban et al.

Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit

Nature

(1999)

J.H. Cate et al.

X-ray crystal structures of 70S ribosome functional complexes

Science

(1999)

Cited by (50)

Eukaryotic peptide chain release factor 1 participates in translation termination of specific cysteine-poor prolamines in rice endosperm
2019, Plant Science
Citation Excerpt :
These sequences have been highly conserved and are essential for the termination of translation [37]. The ESP1/eRF1 protein identified in the present study belongs to the group of eukaryotic class-1 polypeptide chain release factors, the members of which interact with one of three stop codons (UGA, UAG, or UAA) to promote hydrolysis of the ester bond between nascent polypeptide chains and tRNA, thus releasing protein [38,39]. They act in conjunction with class-2 RFs (eRF3 in eukaryotes), which stimulate eRF1 activity in the presence of GTP [38,40].
Prolamines are alcohol-soluble proteins classified as either cysteine-poor (CysP) or cysteine-rich (CysR) based on whether they can be alcohol-extracted without or with reducing agents, respectively. In rice esp1 mutants, various CysP prolamines exhibit both reduced and normal amounts of isoelectric focusing bands, indicating that the mutation affects only certain prolamine classes. To examine the genetic regulation of CysP prolamine synthesis and accumulation, we constructed a high-resolution genetic linkage map of ESP1. The ESP1 gene was mapped to within a 20 kb region on rice chromosome 7. Sequencing analysis of annotated genes in this region revealed a single-nucleotide polymorphism within eukaryotic peptide chain release factor (eRF1), which participates in stop-codon recognition and nascent-polypeptide release from ribosomes during translation. A subsequent complementation test revealed that ESP1 encodes eRF1. We also identified UAA as the stop codon of CysP prolamines with reduced concentration in esp1 mutants. Recognition assays and microarray analysis confirmed that ESP1/eRF1 recognizes UAA/UAG, but not UGA. Our results provide convincing evidence that ESP1/eRF1 participates in the translation termination of CysP prolamines during seed development.
Biotechnology techniques for the development of new tumor specific peptides
2011, Methods
Citation Excerpt :
The diversity of the previously described NEB library PH.D.12™ is diminished from the theoretical value of 4.1 × 1015 to 109 [68]. In ribosome display it is even possible to increase the given theoretical diversity of the library by using error prone-PCR steps or related techniques like DNA shuffling for amplification [69–72]. Every display system is based upon the physical linkage of the phenotype and the genotype of the peptides, miniproteins or antibodies encoded by the library.
Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.
Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.
Single Molecule Studies of Prokaryotic Translation
2008, Single Molecule Biology
This chapter presents an overview of the single molecule methods that have been used for investigating translation and discusses the key discoveries from these methods on the dynamic nature of protein biosynthesis. Single molecule techniques, including fluorescence and force-based methods, provide powerful tools to investigate the multistep and multicomponent process of translation, allowing observation of distance changes on the sub-nanometer scale and the measurement of molecular forces in the piconewton range. Using single molecule fluorescence to monitor the dynamic conformational rearrangements between P-site tRNA and incoming aminoacyl-tRNA has made possible the observation of codon–anticodon sampling and unsuccessful attempts at GTPase activation during initial tRNA selection. Optical trapping methods have recently been applied in the study of translation, yielding insight into the origins of ribosomal motion. Single molecule total internal reflection-based fluorescence resonance energy transfer (TIR-FRET) measurements are particularly useful in the characterization of dynamic conformational changes and to study many key events in translation. Optical tweezer-based force measurements have determined the strength of the mRNA–ribosome complex at various states during translation. These observations have linked peptide bond formation with the weakening of the mRNA–ribosome interaction, which may be necessary for rapid and accurate ribosome movement along the mRNA during translocation. Finally, observing the synthesis of single proteins in vivo has highlighted directly the stochastic nature of gene expression.
mtRF1a Is a Human Mitochondrial Translation Release Factor Decoding the Major Termination Codons UAA and UAG
2007, Molecular Cell
Citation Excerpt :
Once the translation complex has reached a termination codon, the completed protein must be dissociated from the final tRNA, the ribosome, and its cognate mRNA. The proteins responsible for fulfilling these functions are the release factors (RFs), of which there are two classes (for review, see Kisselev et al., 2003; Poole and Tate, 2000). Class I is codon specific with peptide motifs that distinguish the STOP codons.
Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized—for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.
Effect of energy source on the efficiency of translational termination during cell-free protein synthesis
2005, Biochemical and Biophysical Research Communications
We studied how the fidelity of translation termination is affected by the method of ATP regeneration during cell-free protein synthesis. During the in vivo expression of hEPO, whose termination is directed by the UGA codon, we found that substantial proportions of the translational products showed a larger molecular weight than expected. Similar results were obtained in a cell-free synthesis reaction using phosphoenol pyruvate (PEP) or 3-phosphoglycerate (3PG) for ATP regeneration. However, when the energy source was switched to creatine phosphate (CP), the readthrough of the UGA codon was completely repressed and only the target protein of the correct size was expressed in a high yield. To the best of our knowledge, this is the first report describing the relationship between the regeneration of nucleotide triphosphates and protein readthrough, and we also believe that the discovery would pave the way to the selective and efficient expression of target proteins in cell-free protein synthesis systems.
Comparative analysis of base biases around the stop codons in six eukaryotes
2005, BioSystems
Using full-length cDNA sequences, a comparative analysis of sequence patterns around the stop codons in six eukaryotes was performed. Here, it was showed that the codon immediately before and after the stop codons (defined as −1 codon and +1 codon, respectively) were much more biased than other examined positions, especially at the second position of −1 codons and the first position of +1 codons which were rich in As/Us and purines, respectively, for most species. The author speculated that strongly biased sequence pattern from position −2 to +4 might act as an extended translation termination signal. Translation termination was catalyzed by release factors that recognized the stop codons. The multiple amino acid sequence alignment of eukaryotic release factor 1 (eRF1) of 20 species showed that there were 16 residue sites that were strictly conserved, especially the invariant amino acids Ile70 and Lys71. Accordingly, it could be inferred that those candidate amino acids might involve in the recognition process. Moreover, the possible stop signal recognition hypothesis was also discussed herein.

View all citing articles on Scopus

View full text

ReviewRelease factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis

Abstract

Introduction

Section snippets

Structural mimicry among translational complexes

Functional domains of the class I RFs

Fidelity of polypeptide release

Possible mechanisms for translation termination fidelity

Acknowledgments

Cell

Cell

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Biochimie

FEBS Lett.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Mol. Biol.

J. Biol. Chem.

The ribosome returns

Nature

Vizualisation of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation

Proc. Natl. Acad. Sci. USA

Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution

Nature

The small ribosomal subunit from Thermus thermophilus at 4.5 Å resolution: pattern fittings and the identification of a functional site

Proc. Natl. Acad. Sci. USA

Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit

Nature

X-ray crystal structures of 70S ribosome functional complexes

Science

Review
Release factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis