Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes.

An increasing number of cases where tri-nucleotide stop codons do not signal the termination of protein synthesis are being reported. In order to identify what constitutes an efficient stop signal, we analysed the region around natural stop codons in genes from a wide variety of eukaryotic species and gene families. Certain stop codons and nucleotides following stop codons are over-represented, and this pattern is accentuated in highly expressed genes. For example, the preferred signal for Saccharomyces cerevisiae and Drosophila melanogaster highly expressed genes is UAAG, and generally the signals UAA(A/G) and UGA(A/G) are preferred in eukaryotes. The GC% of the organism or DNA region can affect whether there is A or G in the second or fourth positions. We suggest therefore, that the stop codon and the nucleotide following it comprise a tetra-nucleotide stop signal. A model is proposed in which the polypeptide chain release factor, a protein, recognises this sequence, but will tolerate some substitution, particularly A to G in the second or third positions.     

The translational termination signal database.

The Translational Termination Database (TransTerm) consists of the immediate context sequences around the natural termination codons from 45 organisms, and summary tables. The influence of termination codon context on their effectivness as stop signals has been widely documented. The SPECIES--TRI.DAT table shows trinucleotide stop codon usage in each organism and for comparison the occurrence of these sequences in the noncoding region. The SPECIES--TETRA.DAT table contains is a similar table of tetranucleotide stop signal usage. The database is available from EMBL.     

Studies on the bacteriophage MS2. XVI. The termination signal of the A protein cistron

The RNA of bacteriophage MS2 codes for three viral proteins: the coat protein, the A protein and the replicase. Upon infection of various amber suppressor strains of Escherichia coli, we found a fourth viral protein, the synthesis of which was specifically dependent on the presence of an amber suppressor gene. It is shown that this polypeptide is formed by reading through the natural termination signal of the A protein cistron. This cistron therefore terminates with the nonsense codon UAG. The observed prolongation accounts for the addition of some 30 amino acids. Unlike the normal A protein, the longer polypeptide is probably not incorporated into mature phage particles.     

The fate of membrane-bound ribosomes following the termination of protein synthesis

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.     

Asynchronous termination of plasma membrane protein synthesis in erythroid cells.

In this study we focused our attention on the terminal stages of cellular differentiation and asked the question whether the turning off of gene activity is via a general mechanism whereby all proteins are turned off synchronously, or if it is regulated specifically. We examined this problem by measuring the relative rates of synthesis of the plasma membrane proteins in cells that are near the final stages of erythroid differentiation. Our results show that although the rates of synthesis of all proteins decline during maturation the relative rates of decline are different among the various membrane proteins, indicating that the termination of plasma membrane protein synthesis during terminal differentiation is asynchronous.     

Dissecting the energetics of protein alpha-helix C-cap termination through chemical protein synthesis

The alpha-helix is a fundamental protein structural motif and is frequently terminated by a glycine residue. Explanations for the predominance of glycine at the C-cap terminal portions of alpha-helices have invoked uniquely favorable energetics of this residue in a left-handed conformation or enhanced solvation of the peptide backbone because of the absence of a side chain. Attempts to quantify the contributions of these two effects have been made previously, but the issue remains unresolved. Here we have used chemical protein synthesis to dissect the energetic basis of alpha-helix termination by comparing a series of ubiquitin variants containing an L-amino acid or the corresponding D-amino acid at the C-cap Gly35 position. D-Amino acids can adopt a left-handed conformation without energetic penalty, so the contributions of conformational strain and backbone solvation can thus be separated. Analysis of the thermodynamic data revealed that the preference for glycine at the C' position of a helix is predominantly a conformational effect.     

mRNA degradation in procaryotes.

The fast turnover of mRNA permits rapid changes in the pattern of gene expression. In procaryotes, many enzymes involved in mRNA degradation have been identified and some of these endo- and exo-ribonucleases are now being intensively studied. Some of the structural features of mRNA that influence decay rates have also recently been defined. Although important components of the decay pathway are still elusive, a coherent and simple model for mRNA decay has emerged in the last few years.     

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation.

We investigated the cis-acting sequences involved in termination of vesicular stomatis virus mRNA synthesis by using bicistronic genomic analogs. All of the cis-acting signals necessary for termination reside within the first 13 nucleotides of the 23-nucleotide conserved gene junction. This 13-nucleotide termination sequence at the end of the upstream gene comprises the tetranucleotide AUAC, the tract containing seven uridines (U7 tract), and the intergenic dinucleotide (GA), but it does not include the downstream gene start sequence. Data presented here show that upstream mRNA termination is independent of downstream mRNA initiation. Alteration of any nucleotide in the 13-nucleotide sequence decreased the termination activity of the gene junction and resulted in increased synthesis of a bicistronic readthrough RNA. This finding indicated that the wild-type gene junction has evolved to achieve the maximum termination efficiency. The most critical position of the AUAC sequence was the C, which could not be altered without complete loss of mRNA termination. Reducing the length of the wild-type U7 tract to zero, five, or six U residues also totally abolished mRNA termination, resulting in exclusive synthesis of the bicistronic readthrough mRNA. Shortening the wild-type U7 tract to either five or six U residues abolished VSV polymerase slippage during readthrough RNA synthesis. Since neither the U5 nor U6 template was able to direct mRNA termination, these data imply that polymerase slippage is a prerequisite for termination. Evidence is also presented to show that in addition to causing polymerase slippage, the U7 tract itself or its poly(A) product constitutes an essential signal for mRNA termination.     

Trichodermin,a possible inhibitor of the termination process of protein synthesis

The mode of action of the antibiotic, trichodermin, on yeast cells has been investigated. Trichodermin specifically inhibits protein synthesis and, during the in vivo inhibition of protein synthesis, ribosomes remain in polyribosomes rather than shifting to monoribosomes. This observation suggests that trichodermin inhibits either an elongation step or a termination step of protein biosynthesis. These two possibilities were distinguished by comparing the action of trichodermin with that of cycloheximide, a known elongation inhibitor, upon the reformation of polyribosomes during recovery from a block in polypeptide chain initiation. Cycloheximide slows the recovery of polyribosomes from monoribosomes following a block in polypeptide chain initiation whereas trichodermin enhances the recovery of polyribosomes. This observation is interpreted to mean that trichodermin primarily inhibits the termination step of protein biosynthesis.     

Tandem termination signal in plant mRNAs

It was proposed that if some mRNA characteristics resulted in a low efficiency of termination signal, an additional closely located stop codon (tandem stop codons) could be used to prevent the harmful readthrough. However, the role of tandem terminators in higher eukaryotes was not verified and remains hypothetical. In this work the sequence features of Arabidopsis thaliana and Oryza sativa mRNAs were analyzed. It was found that plant mRNAs with UGA terminator were characterized by a higher frequency of nonsense codons in the first triplet position of 3′-UTR that could result from a weak natural selection for “reserve” stop signal. Interestingly, the presence of tandem stop codons positively correlated with a specific amino acid composition in the C-terminal position of the encoded proteins. In particular, C-terminal glycine positively correlated with significantly higher frequencies of reserve terminators at the beginning positions of 3′-UTR in UGA-containing mRNAs. This finding coincides with some earlier observations concerning the role of glycine and its codons in inefficient termination of translation and recoding (e.g., 2A oligopeptide).     

A model for how ribosomal release factors induce peptidyl-tRNA cleavage in termination of protein synthesis

A major unresolved question in messenger RNA translation is how ribosomal release factors terminate protein synthesis. Class 1 release factors decode stop codons and trigger hydrolysis of the bond between the nascent polypeptide and tRNA some 75 A away from the decoding site. While the gross features of the release factor-ribosome interaction have been revealed by low-resolution crystal structures, there is no information on the atomic level at either the decoding or peptidyl transfer center. We used extensive computer simulations, constrained by experimental data, to predict how bacterial release factors induce peptide dissociation from the ribosome. A distinct structural solution is presented for how the methylated Gln residue of the universally conserved GGQ release factor motif inserts into the ribosomal A site and promotes rapid reaction with the peptidyl-tRNA substrate. This model explains key mutation experiments and shows that the ribosomal peptidyl transfer center catalyzes its two chemical reactions by a common mechanism.     

Expression of bacteriophage M13 DNA in vivo. Localization of the transcription initiation and termination signal of the mRNA coding for the major capsid protein.

During the infection cycle of the filamentous bacteriophage M13 a phage specific RNA species is made which selectively directs in vitro the synthesis of the precursor of the major capsid protein encoded by gene VIII. This RNA is unstable (its mean half-life is 11 min) and is made in amounts representing at least 2% of the newly synthesized RN. Nucleotide sequence analysis have indicated that the synthesis of this RNA species is initiated and terminated at the same promoter (G0.18) and termination signal (T0.25) of the M13 genome as the 8S RNA species made in vitro under the direction of M13 replicative form DNA.