Nonsense codon readthrough and changed translational reading frame were measured in different growth phases in E. coli. The strains used carry plasmid constructs with a translation assay reporter gene. This reporter gene contains an internal stop codon or a run of U-residues. Termination or frameshifting give rise to stable proteins that can be physically quantified on gels along with the complete protein products. Readthrough of the stop codon UGA by a nearcognate tRNA is several fold higher in active growth than in late exponential phase. In early exponential phase, about 7% of 31 frameshift at a U W slippery sequence is detectable; upon entry to stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase. A similar increase is observed in the case of +1 reading frameshift at the U W sequence, which increases from 13% in early exponential growth phase up to 38% at the beginning of stationary phase followed by a decrease. Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.z 1998 Federation of European Biochemical Societies.

Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C 1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD + and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD + synthesis is decreased at 30 6C but the level is still sufficient to support normal growth. At 42 6C, NAD + synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD + levels affects ability to grow on minimal medium at 42 6C, while a large decrease leads to a more pleiotropic phenotype.

BackgroundAdenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD+ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD+, especially for one of the alleles, nadD72.ResultsIn this study these two mutant proteins have been further characterized together with ten new mutant variants. Of the, in total, twelve mutations four are in a conserved motif in the C-terminus and eight are in the active site. We have tested the activity of the enzymes in vitro and their effect on the growth phenotype in vivo. There is a very good correlation between the two data sets.ConclusionThe mutations in the C-terminus did not reveal any function for the conserved motif. On the other hand, our data has lead us to assign amino acid residues His-19, Arg-46 and Asp-109 to the active site. We have also shown that the nadD gene is essential for growth in E. coli.

A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.

Thylakoid membranes of pea were used to study competition between extra-membrane fragments and their parental membrane-bound proteins. Phosphorylated and unphosphorylated fragments of light harvesting complex II (LHC II) from higher plants were used to compete with LHC II for interactions with itself and with other thylakoid protein complexes. Effects of these peptide fragments of LHC II and of control peptides were followed by 80 K chlorophyll fluorescence spectroscopy of isolated thylakoids. The phosphorylated LHC II fragment competes with membrane-bound phosphoproteins in the phosphatase reaction. The same fragment accelerates the process of dark-to-light adaptation and decreases the rate of the light-to-dark adaptation when these are followed by fluorescence spectroscopy. In contrast, the non-phosphorylated LHC II peptide does not affect the rate of adaptation but produces results consistent with inhibition of formation of a quenching complex. In this quenching complex we propose that LHC II remains inaccessible to the LHC II kinase, explaining an observed decrease in LHC II phosphorylation in the later stages of the time-course of phosphorylation. The most conspicuous protein which is steadily phosphorylated during the time-course of phosphorylation is the 9 kDa (psbH) protein. The participation of the phosphorylated form of psbH in the quenching complex, where it is inaccessible to the phosphatase, may explain its anomalously slow dephosphorylation. The significance of the proposed complex of LHC II with phospho-psbH is discussed.