Initiation and termination of phage f1 plus-strand synthesis.

The origin of DNA replication of bacteriophage f1 contains a nucleotide sequence that is used both for the initiation of viral (plus) strand synthesis and for its termination. With chimeric plasmids containing two f1 functional origins in the same orientation, synthesis of chimeric plus-strand DNA is initiated, after f1 infection, at either one of the two f1 origins and is terminated at the other. Thus, the chimeric plasmids segregate into two replicons, each of them containing only one f1 origin. This system has been used to test several fragments of the f1 origin varying in size or in nucleotide sequence for their ability to function in either initiation or termination of viral strand synthesis. Our data show that the f1 origin is composed of two overlapping but distinct domains (signals), one for initiation and the other for termination of plus-strand synthesis.     

Initiation of protein synthesis in bacteria at a translational start codon of mamalian cDNA: effects of the preceding nucleotide sequence.

Plasmids containing a mouse cDNA sequence encoding the enzyme dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been used to study the efficiency of initiation of protein synthesis at an ATG (AUG) translational start codon indigenous to the eukaryotic CDNA. differences in DHFR production assayed phenotypically, enzymatically, and immunologically were correlated with the primary structure of the DNA segment that precedes the translational start codon. Our results indicate that initiation of a structurally discrete and biologically functional eukaryotic protein can occur in bacteria on a fused mRNA molecule, and that the efficiency of expression is strongly affected by: (i) the extent of homology of the translational control region with the 3'-OH end of 16S ribosomal RNA, and (ii) the distance between the protein start codon and the ribosome-binding sequence on the mRNA.     

Initiation codon mutation in an Asian Indian family

The beta-thalassemias are a heterogeneous group of hereditary anemias. A multitude of mutations have been reported, resulting in varied phenotypes. In each ethnic group there is always a subset of common, less common, and rare mutations, which makes population screening, prenatal diagnosis, and genetic counseling easier. In this paper we report a rare beta-thalassemia mutation found in an Indian subject by SSCP and sequencing analysis. The mutation, initiation ATG --> ACG, was found in heterozygous condition in a patient belonging to Brahmin family of Uttar Pradesh origin. Haplotype analysis was performed to identify the chromosomal background associated with the mutation and to tracing the origin and spread of the mutation. This study, as previous studies, suggests that rare beta-thalassemia mutations, such as the initiation codon mutations, have no set geographical distribution and are relatively recent.     

Initiation of HeLa cell DNA synthesis in a subnuclear system.

Mammalian cells are known to synthesize DNA in discrete stages, the first of which seems to be the formation of DNA pieces 150--200 nucleotides in length that have a s20 value of about 4 S. We have reconstructed a system derived from HeLa cell nuclei that carries out RNA-primed initiation of the synthesis of small (4S) DNA fragments. This synthesis is resistant to high concentrations of alpha-amanitin and sensitive to antibody directed against RNA polymerase I, suggesting that this enzyme may be involved in the initiation step. The formation of small DNA fragments in this system also requires DNA polymerase alpha, heat-labile nuclear factor(s), and at least one other nuclear protein.     

Reinitiation of translation from the triplet next to the amber termination codon in the absence of ribosome-releasing factor.

Ribosome releasing factor (RR factor) releases ribosomes from mRNA at the termination codon in Escherichia coli. In the absence of this factor, polypeptides with molecular weights very close to the molecular weight of bacteriophage R17 coat protein were synthesized in vitro under the direction of a mutant R17 phage RNA having an amber mutation at codon 7 of the coat cistron. The major coat-protein-like product shared all the R17 coat protein sequence except that the seven NH2-terminal amino acids were missing. The minor product had the complete coat protein sequence starting from formylmethionine except for a probable amino acid substitution at codon 7 (UAG). Addition of RR factor inhibited the synthesis of the major protein. These results indicate that, in the absence of RR factor, the ribosome that has released the NH2-terminal hexapeptide at the amber codon stays on the mRNA and subsequently reinitiates translation "in phase" immediately after the amber codon without formylmethionine.     

Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon.

Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon. Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling techniques. The nucleotide sequence was found to be pG-G-U-U-C-C-A-U-A-m2G-U-A-psi-A-G-D-G-G-D- D-A-G-U-A-C-U-G-G-G-G-A-Cm-U-Um-U-A-i6A-A-psi-C-C-C-U-U-G-A-C- m5C-U-G-G-G-U-psi-C-G-m1A-A-U-C-C-C-A-G-U-G-G-G-A-C-C-U-C-C-AOH. This tRNA sequence exactly matched the DNA sequence of the corresponding tRNA gene. The first position of anticodon is 2'-O-methyluridine (Um), forming UmUA as the anticodon, which presumably recognizes the ochre termination codon UAA. This tRNA species is aminoacylated with glutamine by a Tetrahymena crude aminoacyl tRNA synthetase fraction, suggesting that ochre termination codon is used as a glutamine codon during cytoplasmic protein synthesis in Tetrahymena.     

Translational termination efficiency in mammals is influenced by the base following the stop codon.

The base following stop codons in mammalian genes is strongly biased, suggesting that it might be important for the termination event. This proposal has been tested experimentally both in vivo by using the human type I iodothyronine deiodinase mRNA and the recoding event at the internal UGA codon and in vitro by measuring the ability of each of the 12 possible 4-base stop signals to direct the eukaryotic polypeptide release factor to release a model peptide, formylmethionine, from the ribosome. The internal UGA in the deiodinase mRNA is used as a codon for incorporation of selenocysteine into the protein. Changing the base following this UGA codon affected the ratio of termination to selenocysteine incorporation in vivo at this codon: 1:3 (C or U) and 3:1 (A or G). These UGAN sequences have the same order of efficiency of termination as was found with the in vitro termination assay (4th base: A approximately G >> C approximately U). The efficiency of in vitro termination varied in the same manner over a 70-fold range for the UAAN series and over an 8-fold range for the UGAN and UAGN series. There is a correlation between the strength of the signals and how frequently they occur at natural termination sites. Together these data suggest that the base following the stop codon influences translational termination efficiency as part of a larger termination signal in the expression of mammalian genes.     

Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon.

We have obtained the nucleotide sequence of the genomic RNAs of two alphaviruses, Sindbis virus and Middelburg virus, over an extensive region encoding the nonstructural (replicase) proteins. In both viruses in an equivalent position an opal (UGA) termination codon punctuates a long otherwise open reading frame. The nonstructural proteins are translated as polyprotein precursors that are processed by posttranslational cleavage into four polypeptide chains; the sequence data presented here indicate that the COOH-terminal polypeptide, ns72, may be produced by read-through of this opal codon. The high degree of amino acid homology between the ns72 polypeptides of the two viruses, in contrast to the lack of conserved sequence upstream from the read-through site, suggests that ns72 plays an important role in viral replication, possibly modulating the action of other replicase components.     

Initiation sites of protein folding by NMR analysis.

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.     

Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination.

Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.