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PubMed1981

Usage of the three termination codons: compilation and analysis of the known eukaryotic and prokaryotic translation termination sequences.

The published translation termination sequences have been compiled and analysed to aid the interpretation of experiments on termination codon usage in the Xenopus oocyte (Bienz et al. 1981). There are significant differences between prokaryotes and eukaryotes concerning the usage of the three termination codons and of tandem stops. In addition viruses show termination strategies that differ from those of their hosts. Preferred context sequences flanking termination codons are described. Contexts vary within the last codon according to the nature of the termination codon, but are uniform within the first triplet following the terminators.

Animals↗
PubMedDate not supplied

Correlates of spiritual well-being in terminally ill persons with AIDS and terminally ill persons with cancer.

In an effort to determine if terminally ill patients with AIDS had greater religious and spiritual care needs than other terminally ill patient populations, particularly those with cancer, a study was conducted in a community-based hospice in the southeast. The purpose of the study was to compare the perceptions of spiritual well-being, loneliness, social support, health hardiness, pain, and functional status among terminally ill clients with cancer and terminally ill clients with AIDS in a hospice setting and to examine predictors of spiritual well-being in a hospice population. A sample of 55 hospice patients completed the Correlates of Spiritual Well-Being Scale (COSWEB), which includes a demographic data sheet and instruments to measure spiritual well-being, loneliness, health hardiness, social support, functional status, and pain. Patients with AIDS reported significantly lower spiritual well-being than did patients with cancer and other chronic, terminal illnesses. Patients with AIDS also reported significantly greater loneliness than other patient populations. The number of social supports for patients with AIDS was significantly lower than for cancer patients and other groups; moreover, patients with AIDS were significantly more dissatisfied with their supports than other patient groups. The best predictors of spiritual well-being in this study were social support and loneliness, which explained 47% of the variance in spiritual well-being. The results of this study suggest differences between specific groups of hospice patients. Patients with AIDS may be less spiritually well than other terminally ill patient populations due to decreased support systems, dissatisfaction with supports, greater feelings of loneliness, younger ages on entry to hospice, fewer family supports, lack of recognized long-term relationships, and related issues such as homophobia, perceived rejection by religious denominations, unstable living environments, economic disadvantages, and less time to process life events/meaning. Findings in this study and similar future studies can better enable health care providers to allocate time and resources to various terminally ill patient populations to achieve higher quality care outcomes in general and greater spiritual well-being in particular.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Terminator element mutations affect both the efficiency and position of RNA polymerase I termination in Schizosaccharomyces pombe.

RNA polymerase I transcripts, purified from Schizosaccharomyces pombe cells, terminate at three sites that precede 'Sal box'-like termination element (TE) sequences. Essential features in these elements were investigated by the in vivo expression of targeted mutations. RNA analyses confirmed a functional significance for two of the elements (Boxes 1 and 3), but indicated that the third, less related, sequence (Box 2) does not function as a termination signal. The results further indicated that the most conserved residues in the two active TEs, as well as adjacent regions, are also most critical to function. Furthermore, some mutations in these elements or in immediately flanking sequences affect not only the efficiency of termination, but also alter the position of termination by as much as 35 nt. Since the element is able to influence the site of termination over a surprisingly long stretch of DNA sequence, these observations suggest that the TE does not act simply as a pause element by fixing the termination factor.

Base Sequence↗
PubMed1993

Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription.

NusG is a transcriptional elongation factor in Escherichia coli that aids transcriptional antitermination by the phage lambda N protein. By using NusG affinity chromatography, we found that NusG binds directly and selectively to termination factor rho. NusG was shown previously to be needed for termination by rho in vivo, and we show here that NusG increases the efficiency of termination by rho at promoter-proximal sites in vitro. The rho026 mutation makes termination by rho less dependent on NusG. It also makes antitermination by N at rho-dependent terminators and the binding of rho to NusG temperature sensitive. Therefore, the interaction of NusG with rho is important both for rho-dependent termination and for antitermination by N at rho-dependent terminators.

Bacterial Proteins↗
PubMed1999

Saccharomyces cerevisiae RNA polymerase I terminates transcription at the Reb1 terminator in vivo.

We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3' end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3' end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies.

Base Sequence↗
PubMed2005

Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants.

An approach in determining the intrinsic termination efficiency (%T) of transcription termination using green fluorescent protein (GFP) mutants was developed. This approach utilizes a cassette vector in which the tested terminator is introduced between two GFP mutant genes: an ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and a blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation represents the termination efficiency of the terminator. E. coli ribosomal RNA operon T1 terminator, phage lambda terminator site R2, E. coli tryptophane attenuater were introduced into the vector, and their transcriptional efficiencies were estimated as 89, 79, and 24%, respectively, showing good agreement with published data.

Base Sequence↗
PubMed1990

Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators.

We have investigated the mechanism of transcription termination by T7 RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. The thr attenuator comprises the following two distinct structural elements: a G + C-rich inverted repeat, which encodes an RNA hairpin structure, and A + T-rich regions, one of which contains a continuous sequence of template deoxyadenosine residues within which the transcription terminates. Fourteen attenuator variants were analyzed and we find that not only the hairpin structure itself but also its sequence influences termination. Furthermore, the formation of a hairpin in the RNA encoded by the A + T-rich regions of the attenuator is not mandatory for termination. A series of seven deletion variants that successively shorten the deoxyadenosine tract in the attenuator template were also analyzed. Results from these experiments indicate that complete readthrough occurs when there are four or fewer deoxyadenosine residues. With 5 template deoxyadenosine residues there is 5% termination increasing to 32% with 8 deoxyadenosines, the value produced by the wild-type attenuator. In addition, a comparison with E. coli RNA polymerase shows that T7 RNA polymerase requires a more perfect region of dyad symmetry and a longer deoxyadenosine tract than does the bacterial enzyme to terminate with maximum efficiency.

Base Composition↗
PubMed1996

Role of Escherichia coli RNA polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor NusA.

The alpha subunit (alpha) of RNA polymerase (RNAP) is critical for assembly of polymerase and positive control of transcription initiation in Escherichia coli. Here, we report that alpha also plays a role in transcription elongation, and this involves a direct interaction between alpha and NusA factor. During in vitro transcription without NusA, alpha interacts with the nascent RNA, as revealed by photocrosslinking. When NusA is present, RNA crosslinks to NusA rather than to alpha. We show that this NusA-RNA interaction is diminished during transcription with an RNAP mutant that lacks the C-terminus of alpha beyond amino acid 235, including the so-called alpha CTD. The absence of alpha CTD also affects NusA's ability to enhance transcription pausing, termination at intrinsic terminators and anti-termination by the phage lambda Q anti-terminator, but not anti-termination by the lambda N anti-terminator. NusA functions are not recovered even when transcription with mutant RNAP is done with excess NusA, a condition which does restore NusA-RNA crosslinking. By affinity chromatography, we show that NusA interacts directly with alpha, and also with beta and beta', but not with mutant alpha. Hence, alpha-NusA interaction is vital for the control of transcript elongation and termination.

Bacterial Proteins↗
PubMed1990

Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2.

An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site. Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor. An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition. An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination. The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified. RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found. If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted. These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon.

Base Sequence↗
PubMed2005

Involvement of human release factors eRF3a and eRF3b in translation termination and regulation of the termination complex formation.

eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.

Amino Acid Sequence↗
PubMed1992

Variation in form and axonal termination in the nucleus of the optic tract of the rat: the medial terminal nucleus input on neurons projecting to the inferior olive.

The nucleus of the optic tract (NOT) and the medial terminal nucleus (MTN) are two primary visual nuclei that take part in circuits sustaining the optokinetic reflex. The morphology of rat NOT cells projecting to the inferior olive (NOT-IO neurons) and their terminal input, specifically terminals originating from the MTN, have been studied in the rat at the light and electron microscopical level. This has been done by means of combined retrograde tracing from the inferior olive and anterograde tracing from the MTN to the NOT. The area containing MTN terminal fibers and the area occupied by NOT-IO neurons has been found to match. This matched distribution provides a more detailed description of the NOT, with possible functional implications. Identified NOT-IO neurons demonstrate considerable variability in their dendritic branching pattern and have been found to include all neuronal cell types described for the NOT. The dendritic branching pattern of NOT-IO cells could be related to the orientation and distribution of the NOT's major afferent fiber systems. NOT-IO neurons receive a variable MTN and retinal input onto their somata, comparable to other cells in the NOT. With exception of the superficial part of the NOT, NOT-IO neurons with the most MTN terminals were found dorsally in areas containing large numbers of MTN terminals. In conclusion, although NOT-IO neurons are uniform with respect to their receptive field properties, they vary considerably with respect to the shape of the cell body, dendritic branching pattern, and terminal input. This means that morphological characteristics of NOT-IO neurons have no predictive value with regard to their receptive field properties.

Animals↗
PubMed1994

The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.

We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin.

Acetylation↗
PubMed2006

Orexin-A is composed of a highly conserved C-terminal and a specific, hydrophilic N-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor.

Orexins-A and B, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX(1)R) and orexin-2 (OX(2)R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and (15)N and (13)C natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III:Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I:Leu16-Ala23 and II:Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX(1)R.

Amino Acid Sequence↗
PubMed1995

Automated carboxy-terminal sequence analysis of polypeptides containing C-terminal proline.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vaporphase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-terminal proline.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetic Anhydrides↗
PubMed2001

Characterization of the cis-acting elements controlling subgenomic mRNAs of citrus tristeza virus: production of positive- and negative-stranded 3'-terminal and positive-stranded 5'-terminal RNAs.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has an approximately 20-kb positive-sense RNA genome with two 5' ORFs translated from the genomic RNA and 10 3' genes expressed via nine or ten 3'-terminal subgenomic (sg) RNAs. The expression of the 3' genes appears to have properties intermediate between the smaller viruses of the "alphavirus supergroup" and the larger viruses of the Coronaviridae. The sgRNAs are contiguous with the genome, without a common 5' leader, and are associated with large amounts of complementary sgRNAs. Production of the different sgRNAs is regulated temporally and quantitatively, with the highly expressed genes having noncoding regions (NCR) 5' of the ORFs. The cis-acting elements that control the highly expressed major coat protein (CP) gene and the intermediately expressed minor coat protein (CPm) gene were mapped and compared. Mutational analysis showed that the CP sgRNA controller element mapped within nts -47 to -5 upstream of the transcription start site, entirely within the NCR, while the CPm control region mapped within a 57 nt sequence within the upstream ORF. Although both regions were predicted to fold into two stem-loop structures, mutagenesis suggested that primary structure might be more important than the secondary structure. Because each controller element produced large amounts of 3'-terminal positive- and negative-stranded sgRNAs, we could not differentiate whether the cis-acting element functioned as a promoter or terminator, or both. Reversal of the control element unexpectedly produced large amounts of a negative-stranded sgRNA apparently by termination of negative-stranded genomic RNA synthesis. Further examination of controller elements in their native orientation showed normal production of abundant amounts of positive-stranded sgRNAs extending to near the 5'-terminus, corresponding to termination at each controller element. Thus, each controller element produced three sgRNAs, a 5'-terminal positive strand and both positive- and negative-stranded 3'-terminal RNAs. Therefore, theoretically CTV could produce 30-33 species of RNAs in infected cells.

Base Sequence↗
PubMed1988

Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals, but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum.

The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.

Animals↗
PubMed1988

Immobilized colipase affinities for lipases B, A, C and their terminal peptide (336-449): the lipase recognition site lysine residues are located in the C-terminal region.

Zonal high-performance affinity chromatography has been used in order to study the interactions between pig isolipases A, B and C and the terminal peptide chain fragment 336-449 of the pig lipase on the one hand, and the homolog colipase bound to the inert LiChrosorb diol support on the other. A mathematical treatment led the to assessment of the dissociation constant of the lipase-colipase complex using isolipases or the terminal peptide as eluted acceptors and colipase as silica-bound ligand (Mahé, N., Léger, C.L., Linard, A. and Alessandri, J.-M. (1987) J. Chromatogr. 395, 511-521). A higher affinity of isolipase B as compared to isolipases A and C towards colipase was observed (KD, respectively, of 0.68, 11 and 12 microM) at pH 6.5. Under the same chromatographic conditions, the terminal peptide chain interacted with the bound colipase (KD 0.70 microM, close to that of isolipase B). The chromatographic behaviors of both native and chemically modified lipase and terminal peptide were very similar. In particular, guanidination of lysine residues of both peptide and isolipase B led to the loss of interactions with colipase. The same result was observed with the peptide preincubated in the presence of increasing amounts of free colipase. Accordingly, it is suggested that, firstly, a preferential association of isolipase B to colipase could take place and, secondly, the colipase recognition site of lipase could be located in the C-terminal region, the conformational structure of the terminal peptide not being affected by the enzymic cleavage and, therefore, being largely independent of the rest of the polypeptide molecule. On the other hand, a lower colipase affinity for isolipases A or C than for isolipase B or the C-terminal peptide could tentatively be attributed to a non-local (distant) disturbing effect of the negatively charged glycan chain, as sialic acid is present in both isoforms A and C. Finally, the present paper confirms and extends earlier studies on lipase-colipase interactions.

Amino Acid Sequence↗
PubMed1976

Accumulation of 5-HT in non-terminal axons after p-chloro-N-methylamphetamine without degeneration of identified 5-HT nerve terminals.

The effect of a single injection of d,1-p-chloro-N-methylamphetamine (PCMA) on 5-hydroxytryptamine (5-HT)- containing neurons in rat brain was investigated using fluorescence histochemical, electron microscopic and biochemical methods. PCMA caused in a dose-dependent manner (from 4.3 mg/kg), an increase of formaldehyde-induced indoleamine (IA) fluorescence in swollen non-terminal axons during the first 6 days and, in contrast, a diminution of IA fluorescence in nerve terminal regions for up to 42 days after treatment. These changes did not appear to be the result of destruction of 5-HT nerve terminals since at all time intervals investigated (12 h to 42 days), the fine structure and frequency of supra-ependymal 5-HT nerve terminals were unaffected. Moreover, no degenerating nerve terminals were observed in the suprachiasmatic nucleus. A marked transient decrease of IA fluorescence on day 2 in the 5-HT cell bodies B3-B9 was not followed by obvious morphological changes up to 42 days after PCMA. Therefore, the reduced 5-HT content of brain up to 42 days after treatment seems not to be due to a destruction of 5-HT neurons. Moreover, the damage to non-terminal 5-HT axons, as indicated by the 5-HT accumulation, seems not to be severe, at least not to those axons projecting to the cerebral ventricles and suprachiasmatic nucleus, since no degeneration of 5-HT nerve terminals was observed at any of the times investigated.

Animals↗