Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.     

Translation initiation: adept at adapting.

Initiation of protein synthesis requires both an mRNA and the initiator methionyl (Met)-tRNA to be bound to the ribosome. Most mRNAs are recruited to the ribosome through recognition of the 5' m7G cap by a group of proteins referred to as the cap-binding complex or eIF4F. Evidence is accumulating that eIF4G, the largest subunit of the cap-binding complex, serves as a central adapter by binding to various translation factors and regulators. Other translation factors also have modular structures that facilitate multiple protein-protein interactions, which suggests that adapter functions are common among the translation initiation factors. By linking different regulatory domains to a conserved eIF2-kinase domain, cells adapt to stress and changing growth conditions by altering the translational capacity through phosphorylation of eIF2, which mediates the binding of the initiator Met-tRNA to the ribosome.     

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.     

Translation termination and yeast prions

Protein biosynthesis is the final step in the transfer of genetic information in the cell. In turn, its last step is the release of a nascent polypeptide from the ribosome. Therefore, termination of translation may be considered (if we do not take into account protein post-translational modification and folding) as a final step of the transition from genotype to phenotype through the classic DNA--RNA--protein pathway. In a narrow sense, termination of translation is the hydrolytic cleavage of peptidyl-tRNA into free tRNA and completed polypeptide chain carrying all the information encoded in the corresponding mRNA and DNA. Then the completed protein molecule is released from the ribosome and the ribosome dissociates into its components (subunits, factors, mRNA, tRNA, etc. ). After the synthesis is completed, the polypeptide chain is folded either cotranslationally or by an additional specialized mechanism, depending on the nature of the protein, organism, and other factors. This issue of Biochemistry (Moscow) highlights from various points of view the problem of translation termination, excluding protein folding. Yeast termination factors with prion-like properties are also considered.     

Computer analysis of regulatory signals in complete bacterial genomes. Translation initiation of ribosomal protein operons]

Signals of translation initiation of operons of Haemophilus influenzae ribosomal proteins were predicted. This process is regulated by the formation of secondary RNA structures to which one of the proteins encoded in a particular operon binds. In some cases, these structures imitate the region of protein binding to rRNA. Predictions are made by comparing with homologous operons of Escherichia coli and analogous regions of rRNA and by estimating the energy of secondary structure formation. It is shown that this regulatory mechanism occurs: in operons L11, S10, S15, spc, and alpha of H.influenzae and, probably, in operon S15 of Helicobacter pylori, Bacillus subtilis, and Mycoplasma genitalium.     

Mutation of a termination codon affects src initiation.

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

Recognition of translational termination signals

Ribosomes can specifically shift at certain codons so that the mRNA is read in two different reading frames. To determine if frameshifting occurs at the level of termination, polymers of defined sequence containing AUG, a coding sequence and an in- or out-of-phase nonsense codon were used to bind a termination substrate or to program synthesis and release of dipeptides in a highly purified in vitro translation system. fMet-tRNA bound to ribosomes with AUGUAA, AUGUAAn, AUGUUU, AUGUUA or AUGUAn was not a substrate for release factor RF-1. In contrast, AUGU1UAA, AUGU3UAAn, AUGU4UAAn, AUGU5UAAn effected RF-1-dependent release of fMet from ribosomes. This suggests that nonsense codons can stimulate release whether they occur in- or out-of-phase. Addition of exogenous UAA and RF-1 stimulated release with all oligonucleotides tested. Propagation restricted the RF-1-dependent recognition of out-of-phase nonsense codons but did not restrict recognition of in-phase UAA in AUGU3UAAn. Release of dipeptides from ribosomes programmed with AUGU4UAAn occurred without EF-G and with a mutant lacking EF-G activity, suggesting that out-of-phase termination can occur prior to translocation outside the ribosomal A-site. We propose that the ribosome X RF complex is required to complete proteins, but is also able to frameshift at a nonsense codon resulting in occasional out-of-phase termination of protein synthesis.     

Translation termination: a ghost ballet?

In the October 5th issue of Cell, clarify the end of translation on a eubacterial mRNA. By elucidating the molecular choreography of class I and class II release factors within the ribosome, they show how the steps around the release of nascent protein are ordered.     

Predicting initiation and termination of atrial fibrillation from the ECG.

Atrial fibrillation is the most common cardiac arrhythmia, affecting more than two million people in the US. Several therapies for patients with atrial fibrillation are available, but methods to help physicians select the optimal therapy for an individual patient are still required. Knowledge of whether a patient with a normal ECG will exhibit atrial fibrillation in the future, as well as whether atrial fibrillation will terminate spontaneously, would be very useful in clinical routine. The paper presents a software system for predicting the initiation and termination of atrial fibrillation from the ECG. The algorithms have been validated on ECGs from several signal databases. Prediction of the initiation of atrial fibrillation was achieved by detecting premature heart beats and analyzing the morphology of their P waves. Prediction of the termination of atrial fibrillation was based on calculation of the major atrial frequency. This frequency has been shown to decrease significantly prior to the termination of atrial fibrillation. Nevertheless, the effect is much less distinct in the large data set used for this study compared to previous studies. The initiation of atrial fibrillation, however, could be correctly predicted in approximately 75% of the data analyzed.     

Translation initiation: structures, mechanisms and evolution

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.     

Medicolegal considerations in the initiation and termination of resuscitation in Canada.

Medicolegal issues in cardiopulmonary resuscitation (CPR) and emergency cardiac care were considered in the United States by the National Conference on Cardiopulmonary Resuscitation in 1985. This paper discusses these issues in the Canadian context. Although there is little legislation or case precedent in Canada to guide providers of CPR in decision-making, there appears to be little risk of liability or prosecution for competently rendered care. Providers should be cautious in withholding or withdrawing resuscitative measures from incompetent patients when brain death has not occurred and cardiovascular unresponsiveness has not been demonstrated. However, resuscitation may be withheld when a competent patient refuses it or if there is another medically and legally valid reason to do so.     

Translation initiation and viral tricks

A variety of viral strategies are utilized for dominance of the host-cell protein synthetic machinery, optimization of viral mRNA translation and evasion of host-cell antiviral responses that act at the translational level. Many viruses exploit regulated steps in the initiation of cellular protein synthesis to their own advantage. They have developed some rather unconventional means for mRNA translation, which were probably adapted from specialized cellular mRNA translation systems. Regardless of the type of translational tricks exploited, viruses typically ensure efficient viral translation, often at the expense of host-cell protein synthesis.