1. Regulation of translation by nascent polypeptide mediated stalling

A number of leader peptides induce translational stalling to regulate translation of a downstream gene. For example, stalling during translation of the tnaC causes the ribosome to block the binding sites for the Rho transcription terminator, leading to transcription and thus translation of the downstream tnaAB genes. Similarly, stalling during translation of secM, mifM, ermCL and catA86L also leads to up-regulation of their respective downstream genes. In these cases, however, the stalled ribosome induces a conformational rearrangement in the mRNA that exposes the Shine-Dalgarno sequence, which in turn allows ribosome binding and subsequent translation of the downstream genes. In contrast, stalling during translation of the eukaryotic upstream open reading frames (uORFs) leads to repression, rather then up-regulation, of the fungal arg2/CPA1 and human cytomegalovirus (hCMV) gp48 genes. The stalling capability of leader peptides can be intrinsic to the peptide sequences, as seen for the SecM, MifM or hCMV gp24 leader peptides, or require additional extrinsic co-effector molecules, such as free amino acids or antibiotics. For example, TnaC stalling requires high levels of free tryptophan, which leads to up-regulation of the tryptophanase TnaA and a tryptophan-specific permease TnaB. Likewise, antibiotic-induced stalling leads to up-regulation of antibiotic resistance genes, for example, erythromycin-induced ErmCL stalling leads to up-regulation of ErmC, a methyltransferase that confers macrolide resistance by modifying the large subunit ribosomal RNA (rRNA). We are interested in structurally analyzing ribosomes stalled during translation of such nascent polypeptide chains in order to understand how the interaction between the nascent chain and the ribosomal tunnel causes translation arrest.