Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

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.

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.

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 tRNATrP as compared with a suppressor fonn of the same tRNA. This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect.

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.

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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.