DNA and RNA nucleotides are ubiquitous molecules that store and transmit genetic information. The emergence of synthetic elements that fulfill the function of DNA and RNA provides an alternative gene expression system. Herein, we demonstrate the gene expression of 4'-thioguanine DNA (dSG DNA) via 4'-thiocytosine RNA (dSC RNA) to give green fluorescent protein (GFPuv) in a single test tube. In replication, transcription, and translation, DNA/RNA polymerases and Escherichia coli (E. coli) ribosome can tolerate the replacement of O4' with S4' in the nucleotide, despite the fact that sulfur has a larger atomic radius than oxygen. Additionally, dSG DNA and dSC RNA acted as alternative genetic polymers to natural DNA and RNA for protein synthesis in artificial cells comprising a reconstituted E. coli gene expression machinery. This work involved simple experiments that are widely used in molecular biology, but which underscore the feasibility of life control by substances other than DNA/RNA nucleotides.