The influence of the 5' codon context on translation termination in Bacillus subtilis and Escherichia coli is similar but different from Salmonella typhimurium.

The last two amino acids in the nascent peptide influence translation termination in E. coli (Mottagui-Tabar et al., 1994; Björnsson et al., 1996). We have compared the effects on termination in Escherichia coli, Bacillus subtilis and Salmonella typhimurium obtained by varying the -1 and -2 codons upstream of the weak UGAA stop signal. The peptide effect from the penultimate amino acid on translation termination in B. subtilis is similar to that seen in E. coli (with 66.5% RF-2 amino acid sequence similarity), whereas the influence in S. typhimurium (with 95.3% similarity to E. coli) is weaker. The effect of changing the -1 codon (P-site) is weaker in S. typhimurium as compared to those in E. coli and B. subtilis. RF-2s from E. coli and S. typhimurium have a threonine or alanine at position 246, respectively. This amino acid exchange in RF-2 can explain the difference in efficiency and toxicity during overexpression when E. coli and S. typhimurium are compared (Uno et al., 1996). However, B. subtilis RF-2 also has an alanine at that position, yet the sensitivity to the nascent peptide is similar to that in E. coli. Thus, the amino acid difference at position 246 in the RF-2 sequences cannot explain why termination in E. coli and B. subtilis is similar in peptide sensitivity while being different from that in S. typhimurium. Sequence alignments of RF-2 from the three bacteria show other regions of the molecule that could be involved in the functional interactions with the C-terminal end of the nascent peptide.

Only the last amino acids in the nascent peptide influence translation termination in Escherichia coli genes.

Efficiency of translation termination is affected if the last two amino acids in the nascent peptide are changed [1,2]. By changing the corresponding codons upstream of the stop signal UGAA, we have analyzed if the -3 to -6 amino acids at the C-terminal region of the nascent peptide also affect termination. Lysine at position -3 gave increased readthrough, whereas a total of 28 variations at positions -4, -5, and -6 showed no significant effect on readthrough. The 3'-ends corresponding to the last six codons in 27 Escherichia coli genes were inserted upstream of a stop codon in the 3A' translation assay gene [1]. Readthrough of the stop codon was measured and a possible correlation with the Codon Adaptation Index (CAI) 131 of the respective genes was investigated. Sequences from genes with low CAI do not give any such correlation, whereas sequences from genes with high CAI values are correlated with high termination efficiency. This correlation disappears if the -1 and -2 codons/amino acids are changed. The results suggest that mainly the terminal dipeptide of the terminal hexapeptide sequence has an influence on termination in the tested E. coli genes. This influence is dependent on the charge of the -2 amino acid and is correlated with the alpha-helix propensity of the -1 amino acid, in accordance with results obtained from synthetic gene constructs [1,2].

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding.

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249-257; Björnsson et al (1996) EMBO J 15, 1696-1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA(Trp) by varying the codons at positions-1 and -2 at the 5' side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J4, 1049-1052). The effect on readthrough by changing these-1 and -2 codons is different on the two forms of tRNA(Trp) and is also dependent on the structure of EF-Tu. Readthrough by the tRNA(Trp)-derived suppressor, but not wild-type tRNA(Trp), is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA(Trp) are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA(Trp) at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509-1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756-3760) were found not be altered in sensitivity to the last two amino acids in the nascent peptide.

Antisense transcription in the mammalian transcriptome.

Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring "genes" in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.

Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons.

An in vivo translation assay system has been designed to measure, in one and the same assay, the three alternatives for a ribosome poised at a stop codon (termination, read-through and frameshift). A quantitative analysis of the competition has been done in the presence and absence of release factor (RF) mutants, nonsense suppressors and an upstream Shine-Dalgarno-like sequence. The ribosomal +1 frameshift product is measurable when the stop codon is decoded by wild-type or mutant RF (prf A1 or prf B2) and also in the presence of competing suppressor tRNAs. Frameshift frequency appears to be influenced by RF activity. The amount of frameshift product decreases in the presence of competing suppressor tRNAs, however, this decrease is not in proportion to the corresponding increase in the suppression product. Instead, there is an increase in the total amount of protein expressed from the gene, perhaps due to the purging of queued ribosomes. Mutated RFs reduce the total output of the reporter gene by reducing the amount of all three protein products. The nascent peptide has earlier been shown to influence the translation termination process by interacting with the RFs. At 42 degrees C in a temperature-sensitive RF mutant strain, protein measurements indicate that the nascent peptide seems to influence the binding efficiencies of the RFs.

The second to last amino acid in the nascent peptide as a codon context determinant.

Forty-two different sense codons, coding for all 20 amino acids, were placed at the ribosomal E site location, two codons upstream of a UGA or UAG codon. The influence of these variable codons on readthrough of the stop codons was measured in Escherichia coli. A 30-fold difference in readthrough of the UGA codon was observed. Readthrough is not related to any property of the upstream codon, its cognate tRNA or the nature of its codon-anticodon interaction. Instead, it is the amino acid corresponding to the second upstream codon, in particular the acidic/basic property of this amino acid, which seems to be a major determinant. This amino acid effect is influenced by the identity of the A site stop codon and the efficiency of its decoding tRNA, which suggests a correlation with ribosomal pausing. The magnitude of the amino acid effect is in some cases different when UGA is decoded by a wildtype form of tRNA(Trp) as compared with a suppressor form of the same tRNA. This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect.

The influence of 5' codon context on translation termination in Saccharomyces cerevisiae.

Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C-terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P-site tRNA is able to influence termination at UGAC in yeast.

A polymorphism in the leptin promoter region (-2548 G/A) influences gene expression and adipose tissue secretion of leptin.

There is a large inter-individual variation in circulating leptin concentrations at each level of body fat content. The reason for this is unknown. We investigated whether a polymorphism in the promoter region of the leptin gene (-2548G/A) influences gene transcription and leptin expression in 39 non-obese female subjects. Eleven subjects were homozygous for the AA genotype, 18 were heterozygous (GA) and 10 carried the GG genotype. AA subjects had higher levels of serum leptin than did GA/GG subjects (14.5 +/- 2.1 vs. 9.7 +/- 0.9 ng/ml, p = 0.02). Adipose tissue leptin secretion rate in AA subjects was twice as high as in GA/GG subjects: 1158 +/- 288 vs. 626 +/- 84 ng/2 h/10 (7) cells (p = 0.02). These differences were also statistically significant with leptin levels adjusted for body mass index (p = 0.03 - 0.04). Adipose tissue leptin mRNA levels were 60 % higher in AA subjects, as compared to GA/GG subjects, 74 +/- 10 vs. 46 +/- 4 amol/ micro g RNA (p = 0.01). EMSA revealed that nuclear extracts derived from both U937 cells and human adipocytes form a protein-DNA complex with the leptin -2548G/A polymorphic site and bind with higher affinity to the -2548A-site. In conclusion, a common polymorphism in the promoter of the human leptin gene (-2548G/A) influences leptin expression, possibly at the transcriptional level, and therefore also adipose secretion levels of the hormone.

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.

Evidence for an important role of perilipin in the regulation of human adipocyte lipolysis.

AIMS/HYPOTHESIS: We investigated the role of the adipocyte-specific protein perilipin for lipolysis in humans. METHODS: Perilipin protein content and lipolysis rates were measured in human subcutaneous fat cells of non-obese (n=10) and obese (n=117) women. Single nucleotide polymorphisms in the perilipin gene were examined in obese subjects. RESULTS: Basal and noradrenaline-induced rates of lipolysis were two to fourfold increased (p<0.01) and perilipin protein content decreased 50% (p=0.005) in adipocytes of the obese women. In subjects matched for body mass index and fat-cell volume, a high rate of lipolysis was associated with a low adipocyte content of perilipin (p=0.01). Adipocyte content of perilipin was inversely correlated with the circulating concentrations of glycerol (r=0.62) and non-esterified fatty acids (n=0.49). A gene polymorphism (rs891460 A/G) in intron 6 was common. In AA subjects basal and noradrenaline induced lipolysis were 50 to 100% times more rapid (p

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.