Nascent peptides that block protein synthesis in bacteria

Abstract

Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate expression of a downstream gene. Using a genetic selection in Escherichia coli, we identified additional nascent peptide motifs that stall ribosomes. Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. In particular, three consecutive Pro codons pose a challenge for elongating ribosomes. The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides. The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.

Fig. 1.

Two-hybrid selection for nascent peptides that stall ribosomes. (A) The lambda cI protein binds to DNA upstream of the HIS3 gene. When ribosomes stall during cI synthesis, a short tag is added to the protein by tmRNA, recruiting an SspB-RNA polymerase fusion protein. In this way, ribosome stalling in cI activates transcription of HIS3, restoring the cell’s ability to synthesize histidine and survive on minimal medium. (B) Four cI constructs were tested for their ability to recruit SspB-RpoA, activate HIS3, and grow on medium lacking histidine: cI translationally fused to the tmRNA tag (cI-tag); cI fused to two known inducers of stalling, Glu-Pro-stop (cI-EP-stop) and an mRNA lacking a stop codon (cI-nonstop); and cI with no stalling motif (cI only).

Fig. 2.

Stalling motifs that block termination (T1–T3) or elongation (E1 and E2). (A) Stalling sites for five motifs were determined by toeprinting assays. Blocks in cDNA synthesis are marked with arrows. The antibiotic thiostrepton traps ribosomes in initiation complexes; bands seen in both treated and untreated lanes are reverse transcriptase artifacts. (B) Key amino acids in each motif are written in bold. The codon labeled in yellow is positioned in the P site of the stalled ribosome. The site of stalling in vivo was confirmed by purifying tagged cI, digesting the protein with trypsin, and determining the mass of the C-terminal peptide (highlighted in blue) by MALDI-MS.

Fig. 3.

Key residues in motifs that block termination. (A) The consensus sequence of each motif was determined by randomizing all 20 codons at 30% per nucleotide and subjecting the resulting library to the two-hybrid selection. (B) Toeprinting analyses of a series of mutants for each motif. The original residues are shown above the line, and the substitutions are given below. A thiostrepton-treated control reaction (TS) is shown in the left lane.

Fig. 4.

Key residues in motifs that block elongation. (A) The consensus sequence of the E1 and E2 motifs was determined through random mutagenesis and reselection. In addition, the nucleotide consensus for the HGPP codons is shown below E2. (B) Toeprinting assays reveal the effects of substitutions in the E1 motif (RxPP) (Left) and the E2 motif (HGPP) (Right). (C) Toeprinting assays of E1 and E2, including the minimal motif alone (4), the minimal motif plus seven additional upstream codons (11), and the full-length construct (FL). Ch1 is a chimera containing the minimal motif of E1 (RSPP) with seven upstream codons taken from E2. Ch2 corresponds to the minimal motif of E2 (HGPP) with seven upstream codons from E1.

Fig. 5.

Pre–steady-state kinetic analysis of ribosome stalling. (A) A ribosome complex containing MDTS-peptidyl-tRNA in the P site and the UAA stop codon in the A site was reacted with RF1 to determine the rate of peptidyl-tRNA hydrolysis. Ala (gray) and Phe (black) mutants were characterized to test the contribution of each residue in the DTS motif. (B) A ribosome complex containing MEP-peptidyl-tRNA or derivatives was reacted with RF1. (C) Peptidyl-transfer rates for the E1 motif were obtained by reacting ribosome nascent chain complexes with excess ternary complex composed of Pro- or Phe-tRNA, EF-Tu, and GTP.

Fig. 6.

EF-P alleviates stalling at polyproline stretches but not other stalling motifs. (A) Toeprinting analyses of Ala substitutions in the FxxYxIWPPP motif and a truncated derivative, WPPP. (B) Analysis of pausing in the 20 PPX motifs. The arrow points to the toeprint with the second Pro codon in the P site and the X codon in the A site. (C) Toeprinting analyses of three endogenous E. coli genes with polyproline stretches: lepA, ligT, and amiB. (D) Various motifs were translated in the presence or absence of purified EF-P; the relevant toeprint is labeled with an arrow. (E) MWPPP and MFQKYGIWPPP were translated in vitro with [³⁵S]-methionine in the presence or absence of EF-P. Peptidyl-tRNA accumulates in stalled ribosomes; it was visualized by SDS/PAGE and autoradiography after 3- or 7-min reactions.

Fig. 7.

Ribosome-profiling data highlight pauses at stalling motifs in vivo. Ribosome density is shown across four genes that contain stalling motifs highlighted in red. Read densities are reported in units of reads per million mapped reads. These analyses are of published datasets of E. coli MG1655 ribosome-profiling experiments (38).