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At least 217 records · Page 12Linked to original sources
PubMed1993

Conformations of peptide fragments comprising the amino-terminal, central, and carboxyl-terminal regions of a membrane-active polypeptide. Build-up of secondary structure in pardaxin.

The conformations of synthetic peptides of different lengths corresponding to the amino-terminal, central, and carboxyl-terminal regions of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE) have been studied by circular dichroism spectroscopy. The peptides GFFALIPKIISSPLF-OMe, GFFALIPKIISSPLFK-OMe corresponding to the amino-terminal region, as well as peptides KIISSPLFKTLLSAV and IISSPLFKTLLSAV corresponding to the central region of the toxin have a marked tendency to adopt helical conformation. Ordered conformation is also discernible in the 8- and 7-residue peptides GFFALIPK-OMe and IISSPLF-OMe. Peptides corresponding to the central segments KTLLSAV, LSAVGSAL, and the carboxyl-terminal segment SSSGEQE, however, exhibit very little secondary structure. The peptide segments that adopt ordered conformation show similar conformation when present in the entire toxin as suggested by proton magnetic resonance data (Zagorski, M. G., Norman, D. G., Barrow, C. J., Iwashita, T., Tachibana, K., and Patel, D. J. (1991) Biochemistry 30, 8009-8017). The observation that peptide segments corresponding to the amino-terminal and central regions of the toxin adopt ordered conformations compared to the carboxyl-terminal segment in isolation as well as in the toxin, indicates a role for these regions in initiating and maintaining ordered conformation of pardaxin.

Amino Acid Sequence↗
PubMed1996

Characterization of yeast U1 snRNP A protein: identification of the N-terminal RNA binding domain (RBD) binding site and evidence that the C-terminal RBD functions in splicing.

The yeast U1A protein is a U1 snRNP-specific protein. Like its human counterpart (hU1A), it has two conserved RNA binding domains (RBDs). The N-terminal RBD is quite different from the human protein, and a binding site on yeast U1 snRNA is not readily apparent. The C-terminal RBD is of unknown function. Using in vivo dimethyl sulfate (DMS) protection of mutant strains, we defined a region in yeast U1 snRNA as the likely U1A N-terminal RBD binding site. This was confirmed by direct in vitro binding assays. The site is very different from its vertebrate counterpart, but its location within yeast U1 snRNA suggests a conserved structural relationship to other U1 snRNP components. Genetic studies and sensitive in vivo splicing measurements indicate that the yeast U1A C-terminal RBD also functions in pre-mRNA splicing. We propose that the N-terminal RBD serves to tether the splicing-relevant C-terminal RBD to the snRNP.

Alkylating Agents↗
PubMed1999

C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction.

At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosome-dependent GTPase. The role of the eRF1-eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts of eRF1 (positions 1-280) and of eRF3 (positions 1477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478-530 and 628-637 of eRF3 and positions 281-305 and 411-415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411-415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase.

Amino Acid Sequence↗
PubMed1999

Terminal exon definition occurs cotranscriptionally and promotes termination of RNA polymerase II.

Analysis of nascent transcription from the human epsilon- and beta-globin genes shows that transcriptional termination occurs within 1.5 kb of the poly(A) site and is dependent on the presence of functional poly(A) signals. Even so, transcripts that have not been cleaved at the poly(A) site are detected up to the termination region, suggesting that there is a kinetic lag between transcription over the poly(A) signal and its effect on transcriptional termination. Surprisingly, mutation of the splice acceptor (SA) of the beta-globin IVS2 also abolishes transcriptional termination. Our results emphasize the interconnection of transcription and RNA processing by showing that the enhancement of 3' end processing by the terminal splice acceptor occurs cotranscriptionally.

Cell Nucleus↗
PubMed1991

Transcriptional termination between the closely linked human complement genes C2 and factor B: common termination factor for C2 and c-myc?

We have demonstrated, using a combination of nuclear run-off and poly(A) site competition assays, that transcriptional termination occurs between the closely spaced human complement genes, C2 and Factor B, soon after the C2 poly(A) site. A comparison of the C2 termination signal with a functionally similar sequence downstream of the human alpha 2 globin gene reveals that both signals function in an orientation dependent manner, with subfragments of the whole signal displaying partial effects. In the case of the C2 termination sequence a protein binds within it, and is partially responsible for the termination effect. We further demonstrate that the same (or closely related) protein binds to the ME1a1 site in the murine c-myc promoter, which has been implicated in c-myc attenuation. We suggest that the termination/pause sequences positioned downstream of a gene's poly(A) site may constitute the general signals that elicit transcriptional termination in genes transcribed by RNA polymerase II.

Base Sequence↗
PubMed2002

Antibodies directed against an epitope in the N-terminal region of the H4L subunit of the vaccinia virus RNA polymerase inhibit both transcription initiation and transcription termination, in vitro.

The vaccinia virus virion RNA polymerase that is active in early gene transcription contains a unique subunit encoded by the H4L gene. Prior studies demonstrated that this protein is required both for early gene transcription initiation and for transcription termination. Polyclonal antibodies raised against H4L amino acids 1 to 256 prevent both initiation and termination of transcription, in vitro. Pretreatment of the anti-H4L antibody with a H4L fragment containing amino acids 1 to 99 prevents antibody inhibition of both steps, mapping the inhibitory antibody-binding site to this region. A combination of immunoprecipitation and competition studies of antibody binding to wild-type and site-specific mutations of H4L(1-195) mapped the strong epitope to a site that includes Y18. H4L fragments containing an Y18A mutation exhibit diminished ability to block antibody inhibition of transcription initiation and termination. Antibodies inhibit preinitiation complex (PIC) formation but not the activity of preformed PICs, indicating that this region of H4L interacts with one or more factors during active PIC formation. Furthermore, isolated H4L(1-195) directly inhibits PIC activity, supporting this model. Anti-H4L antibody inhibition of transcription termination is only observed in the absence of the essential termination cofactor NPH I. In contrast, antibody inhibition of PIC formation is unaffected by NPH I, demonstrating that the inhibitory antibody and NPH I can bind to H4L at the same time.

Animals↗
PubMed1988

Transcription termination in Escherichia coli. Measurement of the rate of enzyme release from Rho-independent terminators.

The termination/release phase of transcription must involve at least three major steps: cessation of elongation; release of the transcript; and release of the RNA polymerase. We have devised a novel method for measuring the rate of Escherichia coli RNA polymerase release during transcription termination. The method is based on a kinetic analysis of the rate of RNA synthesis during steady-state transcription. Using this method with defined transcription units, we have found that RNA polymerase release occurs rapidly from several rho-independent terminators. Enzyme release from the T7 early terminator occurs within 13(+/- 3) seconds of the cessation of elongation. Neither nusA protein nor supercoiling of the DNA template affects the rate of enzyme release. However, addition of excess sigma factor significantly increases the rate of enzyme recycling during the steady state. Since added sigma factor does not alter the rates of initiation and elongation by E. coli RNA polymerase holoenzyme, it appears that sigma factor stimulates one or more steps in the termination/release process and reduces the rate of enzyme release to a few seconds. We present evidence that suggests sigma may be directly involved in catalyzing release of the core RNA polymerase from the DNA template during transcription termination. The rapid rates of enzyme release we measure make it difficult to be certain of the exact pathway of events that occur in the termination/release phase of transcription. The most plausible pathway involves initial release of the RNA transcript followed by release of core RNA polymerase from the DNA. Studies on the properties of core polymerase-RNA complexes indicate that core polymerase and the RNA transcript probably do not dissociate as a complex from the terminator. Furthermore, these core-RNA complexes are too stable to represent significant intermediates in the termination/release pathway, at least in the early steps of the reaction.

Bacterial Proteins↗
PubMed1987

The remarkable specificity of a new transcription termination factor suggests that the mechanisms of termination and antitermination are similar.

E. coli lysogenic for the temperate, lambda-related phage HK022 do not support lambda growth. The exclusion of lambda is caused by the HK022 nun gene product, which blocks the expression of genes located downstream of and in the same transcription unit as the lambda nutL and nutR sequences. Transcripts terminating prematurely at or near nutR have been detected after inactivation of lambda repressor in lambda, HK022 dilysogens. Nun therefore appears to be a transcription termination factor with a remarkable specificity; it converts the lambda nut sequences, which normally interact with lambda N protein to suppress transcription termination, into terminators. These and other similarities between Nun-promoted termination and N-promoted antitermination argue strongly that the mechanisms of the two reactions have steps in common.

Bacteriophage lambda↗
PubMed1989

Termination of transcription in human mitochondria: identification and purification of a DNA binding protein factor that promotes termination.

In a transcription system using a HeLa cell mitochondrial lysate programmed by mitochondrial DNA constructs containing the main heavy strand promoter and the transcription termination site at the 16S rRNA/leucyl-tRNA boundary, an appreciable fraction of the heavy strand transcripts terminates at this site, as it does in vivo. A DNA binding protein(s) that protects a 28 bp region immediately adjacent and downstream of the 3' ends of the in vivo and in vitro transcripts has been identified in the lysate and highly purified on an oligodeoxynucleotide affinity column. An activity promoting specific termination of heavy strand transcripts copurified with the DNA binding protein(s), pointing to the involvement of this protein in transcription termination. A distinctive modification of the footprint not correlated with terminating activity has also been observed.

Chromatography, Affinity↗
PubMed1997

Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I.

A replication fork barrier (RFB) at the 3' end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3' terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I-specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

3T3 Cells↗
PubMed1990

Ribosomal protein L4 stimulates in vitro termination of transcription at a NusA-dependent terminator in the S10 operon leader.

The 11-gene S10 ribosomal protein operon of Escherichia coli is under the autogenous control of L4, the product of the third gene of the operon. Ribosomal protein L4 inhibits both transcription and translation of the operon. Our in vivo studies indicated that L4 regulates transcription by causing premature termination within the untranslated S10 operon leader. We have now used an in vitro transcription system to study the effect of purified L4 on expression of the S10 operon. We find that the cell-free system reproduces the in vivo observations. Namely, in the absence of L4, most of the RNA polymerases read through the termination site in the S10 attenuator; the addition of L4 results in increased termination at this site. However, RNA polymerase does not terminate at the S10 attenuator, with or without L4, unless an additional factor, protein NusA, is added to the transcription reaction. These results suggest that the attenuator in the S10 operon is a NusA-dependent terminator whose efficiency is regulated by ribosomal protein L4.

Bacterial Proteins↗
PubMed1987

Natural point mutations within rat rDNA transcription terminator elements reveal the functional importance of single bases for factor binding and termination.

The rat rDNA transcription unit extends 560-565 bp into the spacer downstream of the 28S rRNA coding region. The site of 3' end formation is located in front of a conserved 18 bp sequence element which is repeated eight times in the 3' spacer between nucleotides +582 and +1767 relative to the 3' terminus of 28S rRNA. These sequence motifs are almost identical to the RNA polymerase I transcription termination signal (the Sal I box) that has previously been identified in the 3' terminal spacer of mouse rDNA. Interestingly, each of the single rat elements contains one or more base substitutions as compared to the murine Sal I box. Individual rat Sal I boxes were cloned and tested for their ability to interact with the murine termination factor and to direct transcription termination. It is shown that five of the eight boxes represent genuine transcription terminators, while three elements contain certain point mutations which are not recognized by the nuclear Sal I box-binding protein and therefore are functionally inactive.Images

Animals↗
PubMed2002

Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome.

Ribosome recycling factor (RRF), coded for by the frr locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles. In a cross-species-complementation experiment, it was shown that the Thermus thermophilus RRF protein lacking five amino acids from its C-terminal end (deltaC5TthRRF) but not the full-length protein (TthRRF) complemented Escherichia coli for its frr(ts) phenotype. It was also shown that the Mycobacterium tuberculosis RFF protein (MtuRRF) did not complement E. coli LJ14 for frr(ts). However, simultaneous expression of elongation factor G (EFG) and RRF from M. tuberculosis resulted in complementation of E. coli LJ14. Here it is shown that unlike deltaC5TthRRF, an equivalent mutant of MtuRRF lacking six amino acids from its C-terminal end (deltaC6MtuRRF) did not complement E. coli LJ14. Surprisingly, deltaC6MtuRRF failed to complement the strain even in the presence of homologous EFG (MtuEFG). The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly. However, ribosome-binding assays showed that the mutant protein was compromised in its binding to E. coli ribosomes. It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.

Amino Acid Sequence↗
PubMed2005

[A comparison of perceived nursing needs among oncology nurses, patients with non-terminal cancer and patients with terminal cancer].

PURPOSE: The purpose of this study was to compare the perceived importance and the perceived caring of nursing needs among oncology nurses, patients with non-terminal cancer and patients with terminal cancer. METHOD: A total of 83 oncology nurses, 56 patients with non-terminal cancer and 39 patients with terminal cancer served as subjects. Data was collected based on the 4-point Likert scale using a self-administered questionnaire from Mar. to Sept. 2004. Finally, data was analyzed using mean, SD, paired-test, and ANOVA. RESULTS: The score of the perceived importance of nursing needs was higher than that of the perceived performance of nursing needs in all three groups. There was also a difference in the degree of perceived performance of nursing needs among the three groups. In contrast, there was no difference in the total score of the perceived importance of nursing needs among the three groups, unlike the importance of informational and physical needs as a subgroup of perceived importance, where a difference was noted. CONCLUSIONS: Strategies should be developed to narrow down these gaps between nurses and patients. In particular, informational and educational programs should be designed for patients with terminal cancer.

Adult↗
PubMed1975

Termination of transcription in bacteriophage lambda. Heterogeneous, 3'-terminal oligo-adenylate additions and the effects of rho factor.

RNA transcripts were synthesized in vitro from a lambda DNA template with purified Escherichia coli RNA polymerase either in the presence or absence of the protein termination factor, rho. The products were initially characterized by electrophoresis on polyacrylamide slab gels, and two of the lower molecular weight discrete species (6 S and 4 S RNA) were further characterized by standard two-dimensional "fingerprint" analysis. Production of the 4 S RNA was strongly affected by the presence of rho, whereas production of the 6 S RNA species was relatively unaffected by rho. 3'-Terminal oligonucleotide fragments were then selectively isolated on columns of dihydroxyboryl-substituted cellulose from these transcripts. Sequence analysis of these oligonucleotide products indicated: (a) that all of the transcripts examined possess similar degrees of 3'-terminal sequence heterogeneity which consisted predominantly of the addition of 1 to 5 adenylate residues to the 3'-terminus of the transcript; and (b) that rho factor-enhanced termination results in a definite structural change in the nucleotide sequence with which an RNA molecule can terminate.

Adenine Nucleotides↗
PubMedDate not supplied

[Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3].

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.

Animals↗
PubMed1998

Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex.

There have been contrasting reports of whether the positioning of a translational stop signal immediately after a start codon in a single oligonucleotide can act as a model template to support efficient in vitro termination. This paradox stimulated this study of what determines the constraints on the positioning of the components in the termination complex. The mini mRNA, AUGUGAA, was unable to support efficient in vitro termination in contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the stop signal with the decoding factor, release factor 2, was stimulated with ethanol or with nucleotide-free release factor 3, or by using (L11-)-ribosomes which have a higher affinity for release factor 2, or unless the fMet-tRNA was first bound to 30S subunits independently of the mini mRNA. An additional triplet stop codon could restore activity of the mini mRNA, indicating that its recognition was not sterically restrained by the stop signal already within it. This suggests that in an initiation complex an adjoining start/stop signal is not positioned to support efficient decoding by release factor unless it is separated from the start codon. Site-directed crosslinking from mRNAs to components of the termination complex has shown that mRNA elements like the Shine-Dalgarno sequence and the codon preceding the stop signal can affect the crosslinking to release factor, and presumably the orientation of the signal to the factor.

Codon, Terminator↗
PubMed2003

Patient with terminal duplication 3q and terminal deletion 5q: comparison with the 3q duplication syndrome and distal 5q deletion syndrome.

Partial duplication of chromosome 3q is a well-described condition of multiple congenital anomalies and developmental delay that resembles the Brachmann-de Lange syndrome. Similarly, an emerging phenotype of a distal 5q deletion syndrome has recently been described. The combination of both chromosome abnormalities has not been previously described. We report on a child with both a de novo duplication of distal 3q (q27 --> qter) and terminal deletion of 5q (q35.2 --> qter). The patient had facial anomalies, hypoplastic toenails, lymphedema of the dorsum of the feet, type I Chiari malformation, a seizure disorder, and moderate developmental delays. The phenotype is compared and contrasted to the few reports of patients with similar terminal 3q duplications and 5q deletions. Our patient did not have the characteristic phenotype of the 3q duplication syndrome, suggesting that the chromosome region responsible for this phenotype is more proximal than the terminal 3q27 region. In addition, comparison with three other reported cases of terminal 5q35 deletions suggests a possible association of terminal 5q deletions with central nervous system (CNS) structural abnormalities.

Abnormalities, Multiple↗