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At least 109 records · Page 6Linked to original sources
PubMed2000

Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain.

Within the IGF axis, the insulin-like growth factor-binding proteins (IGFBPs) are known to play a pivotal role in cell proliferation and differentiation. Defined proteolysis of the IGFBPs is proposed to be an essential mechanism for regulating IGF bioavailability. The generated IGFBP fragments in part exhibit different IGF-dependent and -independent biological activities. Characterizing naturally occurring forms of IGFBPs in human plasma, we identified both a N- and a C-terminal fragment of IGFBP-4 by means of immunoreactivity screening. As a source for peptide isolation, we used large amounts of human hemofiltrate obtained from patients with chronic renal failure. Purification of the IGFBP-4 peptides from hemofiltrate was performed by consecutive cation-exchange and reverse-phase chromatographic steps. Mass spectrometric and sequence analysis revealed an M(r) of 13 233 for the purified N-terminal fragment spanning residues Asp(1)-Phe(122) of IGFBP-4 and an M(r) of 11 344 for the C-terminal fragment extending from Lys(136) to Glu(237). Proteolytic digestion and subsequent biochemical analysis showed that the six cysteines of the C-terminal IGFBP-4 fragment are linked between residues 153-183, 194-205, and 207-228 (disulfide bonding pattern, 1-2, 3-4, and 5-6). Plasmon resonance spectroscopy, ligand blot analysis, and saturation and displacement studies demonstrated a very low affinity of the C-terminal IGFBP-4 fragment for the IGFs (IGF-II, K(d) = 690 nM; IGF-I, K(d) > 60 nM), whereas the N-terminal fragment retained significant IGF binding properties (IGF-II, K(d) = 17 nM; IGF-I, K(d) = 5 nM). This study provides the first molecular characterization of circulating human IGFBP-4 fragments formed in vivo exhibiting an at least 5-fold decrease in the affinity of the N-terminal IGFBP-4 fragment for the IGFs and a very low IGF binding capacity of the C-terminal fragment.

Amino Acid Sequence↗
PubMed1995

Localization of the N-terminal and C-terminal ends of triadin with respect to the sarcoplasmic reticulum membrane of rabbit skeletal muscle.

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-17) and C-terminal (residues 691-706) ends of rabbit skeletal muscle triadin, a 95 kDa protein located in the sarcoplasmic reticulum membrane at the triad junction. The specificity of the antibodies generated was tested by ELISA and Western blot analysis. These tests demonstrated the ability of the antibodies to react specifically with the proteins. The anti-N-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that the N-terminal end of the membrane-embedded triadin is exposed on the cytoplasmic side of the vesicles. In contrast, the anti-C-terminus antibodies were able to react with sarcoplasmic reticulum vesicles only after permeabilization of the vesicles with a detergent, indicating that the C-terminal end is exposed on the luminal side of the vesicles. These immunological data were complemented by proteolysis experiments using carboxypeptidases and endoproteinase Arg C. A mixture of carboxypeptidases A, B and Y was used to induce degradation of the C-terminal end of triadin in sarcoplasmic reticulum vesicles. This degradation, and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, was observed only when the vesicles were permeabilized, providing further evidence for the luminal localization of the C-terminal end of triadin. Treatment of sarcoplasmic reticulum vesicles with endoproteinase Arg C resulted in the removal of the N-terminal end of triadin, probably due to cleavage after Arg-34. This is a further indication of the cytoplasmic localization of the N-terminal end of triadin (and of its first 34 amino acids). When the proteolysis with endoproteinase Arg C was carried out with permeabilized vesicles, the cleavage occurred after Arg-141 or Arg-157, indicating that at least one of these residues is luminal.

Amino Acid Sequence↗
PubMed1995

SecA-independent translocation of the periplasmic N-terminal tail of an Escherichia coli inner membrane protein. Position-specific effects on translocation of positively charged residues and construction of a protein with a C-terminal translocation signal.

We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G. (1994) EMBO J. 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and found that the introduction of even a single arginine can block translocation. Position-specific differences in the effects on translocation of arginine insertions suggest that the C-terminal end of the N-terminal tail is more critical for translocation than the central and N-terminal regions. We also show that the N-terminal tail is translocated in a truncation mutant where a stop codon is placed immediately after the first transmembrane segment, provided that the transmembrane segment is flanked on its C-terminal end by positively charged residues. Thus, sec-independent translocation of a relatively large domain can be induced by a translocation signal located at the extreme C terminus of a protein.

ATP-Binding Cassette Transporters↗
PubMed2000

Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a "shuttle" peptide to help secretion.

Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N-linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to (1)/(10) that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C-terminal region was found to be important for secretion of TPO. Among six N-glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn-234, were found to be critical for secretion, and two other locations, Asn-319 and Asn-327, did not affect the secretion.

Animals↗
PubMed2001

The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis.

The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.

Adenosine Triphosphatases↗
PubMed2003

UUUUUNU stimulation of vaccinia virus early gene transcription termination. Oligonucleotide sequence and structural requirements for stimulation of premature termination in vitro.

Vaccinia virus early genes are unique in that transcription terminates in a signal- and factor-dependent manner. Recent results from this laboratory demonstrated that a 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence- and concentration-dependent stimulation of premature transcription termination and transcript release in trans. In an effort to better understand the different aspects of the U5NU stimulation of premature termination, we evaluated the activity of various oligonucleotides in vitro. Neither RNA containing a mutant U5NU signal nor single-stranded DNA containing T5NT was able to stimulate premature termination, demonstrating both sequence specificity and a requirement for ribose. Furthermore, neither oligonucleotide was able to compete with U5NU, demonstrating that each failed to bind to the U5NU recognition factor. Substitution of the U9 signal with either BrU9 or BrdU9 inhibited normal termination but did not stimulate premature termination. The addition of BrdU5NdU inhibited U5NU stimulation of premature termination, demonstrating that both oligonucleotides bind to the same site on the U5NU recognition factor. Finally, U5NU containing RNA as short as nine bases served as an effective stimulator of premature termination. These observations impact directly on the development of oligonucleotide based anti-poxvirus therapeutic agents.

Animals↗
PubMed1991

Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils.

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes (PMN) is a potent cytotoxin, specific for Gram-negative bacteria, that also inhibits endotoxin activity by neutralizing isolated bacterial lipopolysaccharides (LPS). We have previously shown that an isolated 25 kD N-terminal fragment of human BPI carries all the antibacterial activities of the parent 55-60 kD molecule. In this study we have compared the LPS-neutralizing activities of human holo-BPI, the N-terminal fragment and a 30 kD C-terminal fragment that we have now isolated. We show that the N-terminal fragment also has LPS-neutralizing activity as detected by inhibition (up to 95%) of (a) activation by LPS of procoagulant proteases in Limulus amebocyte lysates, (b) LPS "priming" of PMN, and (c) LPS-mediated production of tumor necrosis factor in whole human blood. Holo-BPI and the 25 kD fragment have similar neutralizing potency (in nanomolar range) in all assays toward "smooth" LPS from Escherichia coli O111:B4 and O55:B5 (possessing long chain polysaccharide or O-antigen), and "deep rough" LPS from Salmonella minnesota Re595 mutant (possessing no O-antigen). The C-terminal fragment of BPI is devoid of antibacterial activity when tested against BPI-sensitive E. coli J5, but does have endotoxin-neutralizing activity. This activity is weak relative to holo-BPI and the 25 kD N-terminal fragment in the Limulus and PMN-priming assay, but is comparable for inhibition of TNF production in whole blood. We conclude that the principal determinants for LPS recognition and neutralization, like those for antibacterial action, reside in the N-terminal half of the BPI molecule, but that sites within the C-terminal half can also contribute to BPI-LPS interaction once LPS is detached from the bacterial envelope.

Antimicrobial Cationic Peptides↗
PubMed1998

Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3'-end formation and excision of the 3'-terminal intron.

We have analyzed transcription termination, 3'-end formation, and excision of the 3'-terminal intron in vivo in the Balbiani ring 1 (BR1) gene and its pre-mRNA. We show that full-length RNA transcripts are evenly spaced on the gene from a position 300 bp upstream to a region 500-700 bp downstream of the polyadenylation sequence. Very few full-length transcripts and no short, cleaved, nascent transcripts could be observed downstream of this region. Pre-mRNA with 10-20 adenylate residues accumulates at the active gene and then rapidly leaves from the gene locus. Only polyadenylated pre-mRNAs could be detected in the nucleoplasm. Our results are consistent with the hypothesis that transcription termination occurs in a narrow region for the majority of transcripts, simultaneous with 3'-end formation. Excision of the 3'-terminal intron occurs before 3'-end formation in about 5% of the nascent transcripts. When transcription terminates, 3' cleavage takes place and 10-20 adenylate residues are added, the 3'-terminal intron is excised from additionally about 75% of the pre-mRNA at the gene locus. Our data support a close temporal and spatial coupling of transcription termination and the cleavage and initial polyadenylation of 3'-end formation. Excision of the 3'-terminal intron is highly stimulated as the cleavage/polyadenylation complex assembles and 3'-end formation is initiated.

Animals↗
PubMed1975

Terminal-sequence analysis of bacterial ribosomal RNA. Correlation between the 3'-terminal-polypyrimidine sequence of 16-S RNA and translational specificity of the ribosome.

The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequences obtained were: Bacillus stearothermophilus, -G(Z)approximately 5 Y-U-C-C-U-U-U-C-U (A); B. subtilis, -G(Z)approximately 7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aerugionosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B stearothermophilus and B. subtilis, -Y-C; C. crescentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis.

Bacillus subtilis↗
PubMed2000

Initiation and termination of DNA transfer during conjugation of IncI1 plasmid R64: roles of two sets of inverted repeat sequences within oriT in termination of R64 transfer.

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3' terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.

Bacterial Proteins↗
PubMed1996

In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

Amino Acid Sequence↗
PubMed2003

The nonessential H2A N-terminal tail can function as an essential charge patch on the H2A.Z variant N-terminal tail.

Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up approximately 80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

Acetylation↗
PubMed2002

External-loop free energy affects dye-labeled terminators premature terminations in DNA cycle-sequencing reactions.

Although dideoxy terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. I recently demonstrated that combining the annealing step with the extension step at a single temperature (60 degrees C) reduces premature terminations of DNA sequences that ordinarily contain premature terminations when three temperature steps are used in sequencing. I studied a novel class of DNA sequences of 100-bp length and located upstream from the point that causes premature terminations. I determined the thermodynamics of 49 DNA sequences with premature terminations at three temperature steps by using DNA mfold profiles. Sequencing results for 28 samples were improved with two-step cycle sequencing, whereas two-step cycle-sequencing reactions did not improve the results for 21 sequences. Nearest-neighbor thermodynamic parameters for all 49 sequences were compared at temperatures 50 degrees C and 60 degrees C. The parameters predicted that thermodynamic free-base (external-loop) energies (delta delta G degree) were significantly different for these two study groups of samples. The results indicate that changes in free energy in single-strand base (external-loop) sequences can have a significant effect in reducing premature terminations in DNA sequencing reactions run with energy-transfer-based fluorescent-dye terminators.

Base Sequence↗
PubMed1994

Immunolocalization of the C-terminal and N-terminal regions of alpha-A and alpha-B crystallins.

The C-terminal and possibly the N-terminal regions of the alpha crystallins are thought to be involved in the molecular chaperone properties of the protein. To localize these regions within the 13-15 nm aggregate of native alpha crystallin, antisera specific for the C-terminal and N-terminal regions were used together with immunogold and transmission electron microscopy. The results demonstrate that the C-terminal regions of the alpha-A and alpha-B molecules and the N-terminal regions of the alpha-A and/or alpha-B molecules are localized to a central region of the native alpha aggregate. These findings demonstrate that the C-terminal and N-terminal regions of alpha crystallin are localized to a region of the alpha aggregate that has previously been hypothesized to be the binding site for partially denatured proteins.

Animals↗
PubMed2001

Incomplete refolding of a fragment of the N-terminal domain of pig muscle 3-phosphoglycerate kinase that lacks a subdomain. Comparison with refolding of the complementary C-terminal fragment.

Refolding of pig muscle 3-phosphoglycerate kinase (PGK) from a mixture of its complementary proteolytic fragments that did not correspond to the individual domains resulted in a high degree of reactivation [Vas, M., Sinev, M.A., Kotova, N. & Semisotnov, G.V. (1990) Eur. J. Biochem. 189, 575--579]. An independent refolding of the 27.7 kDa C-terminal proteolytic fragment (which encompasses the whole C domain) has been noted, but the refolding ability of the 16.8-kDa N-terminal proteolytic fragment, which lacks a single subdomain from the N domain, remained to be seen. Here the refolding processes of the isolated fragments are compared. Within the first few seconds of initiation of refolding, pulse-proteolysis experiments show the formation of a structure with moderate protease resistance for both fragments. This structure, however, remains unchanged upon further incubation of the N-terminal fragment, whereas refolding of the C-terminal fragment continues as detected by a further increase in proteolytic resistance. The non-native character of the folding intermediate of the N fragment is indicated by the elevated fluorescence intensity of the bound hydrophobic probe 8-anilino-1-naphtalene sulphonate. Its CD spectrum shows the formation of secondary structure distinct from the native one. The noncooperative phase-transition observed in microcalorimetry indicates the absence of a rigid tertiary structure, in contrast with the refolded C-terminal fragment for which a cooperative transition is seen. Size-exclusion chromatography supported the globular character of the intermediate, and showed its propensity to form dimers. No binding of the substrate, 3-phosphoglycerate (3-PGri), to the isolated N-terminal fragment, could be detected but the presence of the complementary C-terminal fragment led to restoration of the substrate binding ability of the N domain. Thus, refolding of the isolated N-terminal fragment yields a highly flexible, globular, potentially productive intermediate with non-native secondary structure and highly exposed hydrophobic clusters, which favour dimerization.

Animals↗
PubMed2000

Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency.

Translation termination is the final step that completes the synthesis of a polypeptide. Premature translation termination by introduction of a nonsense mutation leads to the synthesis of a truncated protein. We report the identification and characterization of the product of the MTT1 gene, a helicase belonging to the Upfl-like family of helicases that is involved in modulating translation termination. MTT1 is homologous to UPF1, a factor previously shown to function in both mRNA turnover and translation termination. Overexpression of MTT1 induced a nonsense suppression phenotype in a wild-type yeast strain. Nonsense suppression is apparently not due to induction of [PSI+], even though cooverexpression of HSP104 alleviated the nonsense suppression phenotype observed in cells overexpressing MTT1, suggesting a more direct role of Hsp104p in the translation termination process. The MTT1 gene product was shown to interact with translation termination factors and is localized to polysomes. Taken together, these results indicate that at least two members of a family of RNA helicases modulate translation termination efficiency in cells.

Adenosine Triphosphatases↗
PubMed1995

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription.

Amino Acid Sequence↗
PubMed1996

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Amino Acid Sequence↗