SUMMARYElectrostatic potentials along the ribosomal exit tunnel are non-uniform and negative. The significance of electrostatics in the tunnel remains relatively uninvestigated, yet is likely to play a role in translation and secondary folding of nascent peptides. To probe the role of nascent peptide charges in ribosome function, we used a molecular tape measure that was engineered to contain different numbers of charged amino acids localized to known regions of the tunnel, and measured chain elongation rates. Positively-charged arginine or lysine sequences produce transient arrest (pausing) before the nascent peptide is fully elongated. The rate of conversion from transiently arrested to full-length nascent peptide is faster for peptides containing neutral or negatively-charged residues than for those containing positively-charged residues. We provide experimental evidence that extra-ribosomal mechanisms do not account for this charge-specific pausing. We conclude that pausing is due to charge-specific interactions between the tunnel and the nascent peptide.

Helicity of membrane proteins can be manifested inside the ribosome tunnel, but the determinants of compact structure formation inside the tunnel are largely unexplored. Using an extended nascent peptide as a molecular tape measure of the ribosomal tunnel, we have previously demonstrated helix formation inside the tunnel. Here, we introduce a series of consecutive polyalanines into different regions of the tape measure to monitor the formation of compact structure in the nascent peptide. We find that the formation of compact structure of the polyalanine sequence depends on its location. Calculation of free energies for the equilibria between folded and unfolded nascent peptides in different regions of the tunnel shows that there are zones of secondary structure formation inside the ribosomal exit tunnel. These zones may have an active role in nascent-chain compaction.

Transmembrane segments in the intact voltage-gated potassium (Kv) channel are helical. To ascertain whether this helicity could first be manifested inside the ribosomal tunnel, we generated biogenic peptide intermediates of Kv1.3 and mass-tagged the cysteine-scanned S6 transmembrane segment using pegylation (PEG-MAL) and calmodulation (CaM-MAL). For reference, we created an extended peptide that was used as a "molecular tape measure" of the ribosomal tunnel and determined that the functional length of the tunnel is 99-112 A. We demonstrate that the S6 segment forms a compact structure inside the ribosomal tunnel and that the N-terminal half of S6 compacts more than the C-terminal half of S6. These results bear on the earliest folding events during biogenesis of ion channels.

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