Eukaryotic DNA is organized and compacted in a string of nucleosomes, DNA-wrapped protein cylinders. The positions of nucleosomes along DNA are not random but show well-known base pair sequence preferences that result from the sequence-dependent elastic and geometric properties of the DNA double helix. Here, we focus on DNA around transcription start sites, which are known to typically attract nucleosomes in multicellular life forms through their high GC content. We aim to understand how these GC signals, as observed in genome-wide averages, are produced and encoded through different genomic regions (mainly 5′ UTRs, coding exons, and introns). Our study uses a bioinformatics approach to decompose the genome-wide GC signal into between-region and within-region signals. We find large differences in GC signal contributions between vertebrates and plants and, remarkably, even between closely related species. Introns contribute most to the GC signal in vertebrates, while in plants the exons dominate. Further, we find signal strengths stronger on DNA than on mRNA, suggesting a biological function of GC signals along the DNA itself, as is the case for nucleosome positioning. Finally, we make the surprising discovery that both the choice of synonymous codons and amino acids contribute to the nucleosome positioning signal.