Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells

Transcription termination and 3' processing: the end is in site!

Although chemists can synthesize virtually any small organic molecule, our ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, modifications are largely restricted to substitutions among the common 20 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and eukaryotic organisms. Over 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive, and redox-active amino acids, glycosylated amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom-containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.

Expanding the genetic code.

A key step in retrieving the information stored in the complex genomes of eukaryotes involves the identification of transcription units and, more specifically, the recognition of promoter sequences by RNA polymerase. In eukaryotes, the task of recognizing nuclear gene promoters and then transcribing the genes is divided among three highly related enzymes, RNA polymerases I, II, and III. Each of these RNA polymerases is dedicated to the transcription of specific sets of genes, and each depends on accessory factors, the so-called transcription factors, to recognize its cognate promoter sequences.

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Recruitment of RNA polymerase III to its target promoters

Recently, a general method was developed that makes it possible to genetically encode unnatural amino acids with diverse physical, chemical, or biological properties in Escherichia coli, yeast, and mammalian cells More than 30 unnatural amino acids have been incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA/aminoacyl-tRNA synthetase pair These include fluorescent, glycosylated, metal-ion-binding, and redox-active amino acids, as well as amino acids with unique chemical and photochemical reactivity This methodology provides a powerful tool both for exploring protein structure and function in vitro and in vivo and for generating proteins with new or enhanced properties

The split promoter sequences of a tRNALeuCUG gene of Xenopus laevis have been mapped to nucleotides 13–20 and 51–64 of the tRNALeu coding sequences. The sequences closely coincide with two conserved sequence blocks present in all eukaryotic tRNA genes. The two conserved sequence blocks were found to be exchangeable between tRNA genes as chimaeric tRNAMet–tRNALeu genes proved transcriptionally active. Furthermore, two prokaryotic tRNA genes exhibiting strong homologies with the two blocks yielded specifie transcripts when tested in an eukaryotic transcriptional system.

Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements

https://www.cell.com/cell/pdf/0092-8674(83)90539-1.pdf

Gabriella Galli

Papers

Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements

Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini.