Surrounding Nucleotide Sequence Research Articles

Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases.

Nuclear magnetic resonance studies on transfer ribonucleic acid: assignment of AU tertiary resonances

The hydrogen-bonded ring NH nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acid (RNA) species have been examined with particular emphasis on the extreme low-field portion. Betwen --13.8 and --15 ppm there are two extra resonances which are not derived from cloverleaf base pairs. A combined approach involving undermodified tRNAs, chemical modification, and hairpin fragment studies has assigned the T54--A58 resonance at --14.3 ppm in yeast tRNAPhe and Escherichia coli tRNA1 Val., the U8--A14 resonance has been assigned at --14.3 ppm, and the s4U8--A14 resonance in bacterial tRNAs has been assigned at --14.9 ppm. The T54--A58 resonance shifts between --14.3. and --13.8 ppm depending on the surrounding nucleotide sequence in the ribothymidine loop.

Inability of circular mRNA to attach to eukaryotic ribosomes.

IN contrast to prokaryotic systems, initiation by eukaryotic ribosomes seems to be restricted to a single site near the 5′-terminus of the message. However, nucleotide sequence analyses have not revealed a common ‘recognition sequence’ within the 5′-proximal portion of eukaryotic messages. We have previously suggested that the position of an AUG codon within a eukaryotic message—rather than the surrounding nucleotide sequence—might be the primary feature defining an initiation site. We proposed1,2 that a 40S ribosomal subunit binds initially at or near the 5′-end of a message and migrates along the RNA chain until the first AUG codon is encountered, whereupon it stops, a 60S subunit joins, and polypeptide synthesis begins. Although this mechanism accounts for many of the peculiarities of eukaryotic translational initiation, it leaves unanswered the question of how ribosomes recognise the 5′-terminus of mRNA. The m7G cap which blocks the end of most eukaryotic messages3 is not, responsible for directing ribosomes to the 5′-terminus since ribosomes initiate at the 5′-proximal AUG even in uncapped or unmethylated RNAs4,5. The ability of ribosomes to select the 5′-end of a message irrespective of its chemical structure (that is, whether the terminus is m7GpppN…, GpppN… or pppN…) is difficult to explain. An intriguing possibility is that eukaryotic ribosomes attach to mRNA by a threading mechanism. A simple prediction of the threading mechanism is that circularisation of a message should abolish its ability to interact with ribosomes. The experiments described below fulfill this prediction. The possibility of threading by prokaryotic ribosomes was ruled out, several years ago, by the demonstration that Escherichia coli ribosomes were able to translate the circular DNA of phage fd6,7. Indeed, the simplest version of a threading mechanism (entry at the 5′-terminus of a message with translation beginning at the first AUG codon encountered by the ribosome) is contradicted by the ability of E. coli ribosomes to initiate at multiple internal sites in poly-cistronic messages, such as bacteriophage R17 RNA. Conversely, the threading mechanism is consistent with the mono-cistronic character of eukaryotic messages.

Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast : I. rev1 mutant strains

Radiation-sensitive strains of the yeast, Saccharomyces cerevisiae, that carry the rev1-1 mutation exhibit greatly reduced frequencies of reversion of a number of auxotrophic mutations (Lemontt, 1971a), much lower frequencies of forward mutation to auxotrophy (Lemontt, 1972) and substantially reduced frequencies of reversion of cyc1-9 (Lawrence and Christensen, 1976), an ochre allele of the structural gene for iso-1-cytochrome c (Stewart et al., 1972), when exposed to ultraviolet light. In contrast, the u. v. -induced reversion of cyc1-131, whose message contains the valine codon GUG in place of the AUG initiation codon (Stewart et al., 1971), is unaffected by the presence of the rev1-1 mutations (Lawrence and Christensen, 1976). We have confirmed this observation using the rev1-1 as well as the rev1-1 mutation and have also identified four other cyc1 alleles out of 15 examined that behave in the same way. It is clear, therefore, that while the REV1 gene function is required for the production of many mutational alterations at this locus, it is not for the production of certain specific events. The reason for this appears to depend on the genetic nature of these events rather than the kind of premutational lesion but, despite almost complete information on the base-pair changes and surrounding nucleotide sequences at these sites, it has not yet been possible to define their special nature precisely, indicating that the mutational process is surprisingly complex. The REV1 gene function is not required for the production of most base-pair additions or deletions and although necessary for the formation of most base-pair substitutions it is also not required for the substitutions that lead to the reversion of cyc1-131 and the proline missense mutant, cyc1-115. The REV1 gene function is necessary to produce identical substitutions at other sites, however, and also to produce different substitutions at the cyc1-131 site, suggesting that the special character of cyc1-115 and cyc1-131 is the requirement for a specific base-pair alteration at a specific site. Whatever the explanation, the evidence of this kind of allele-specific control of u.v. -induced reversion must be accounted for by any proposed model of the mutagenic process and must also be accommodated by any scheme to test environmental mutagens.