Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues.

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest.

The Identity of the Constriction Region of the Ribosomal Exit Tunnel Is Important to Maintain Gene Expression in Escherichia coli.

Mutational changes in bacterial ribosomes often affect gene expression and consequently cellular fitness. Understanding how mutant ribosomes disrupt global gene expression is critical to determining key genetic factors that affect bacterial survival. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which displayed alterations during growth.

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.

Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp.

The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA.

The regulatory gene from the operon of controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp-dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known translation termination factors, RF1 and RF2.

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp.

Inhibition of essential bacterial peptidyl-tRNA hydrolase activity by tropical plant extracts.

Peptidyl-tRNA Hydrolase (Pth) is a highly conserved, essential enzyme in bacteria. It removes the peptide portion from peptidyl-tRNA, returning free tRNAs to participate in translation. Build-up of peptidyl-tRNAs is toxic and defects in Pth function result in cell death.

Peptidyl-tRNA hydrolase screening combined with molecular docking reveals the antibiotic potential of Syzygium johnsonii bark extract.

With the rapid rise of antibiotic resistance in pathogenic bacteria, the need for new antibacterial agents is overwhelming. Herein we report the limited screening of tropical plant extracts for inhibitory activity against the essential enzyme peptidyl-tRNA hydrolase (Pth). Initial screening was conducted through an electrophoretic mobility assay and Northern blot detection.

Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation.

Nascent polypeptide sequences that influence ribosome function.

Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment.

Tryptophan inhibits Proteus vulgaris TnaC leader peptide elongation, activating tna operon expression.

Expression of the tna operon of Escherichia coli and of Proteus vulgaris is induced by L-tryptophan. In E. coli, tryptophan action is dependent on the presence of several critical residues (underlined) in the newly synthesized TnaC leader peptide, WFNIDXXL/IXXXXP.

23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction.

Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA(Pro) leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA(Pro) by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA(Pro) to puromycin.

Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression.

In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression.

Physiological effects of anti-TRAP protein activity and tRNA(Trp) charging on trp operon expression in Bacillus subtilis.

The Bacillus subtilis anti-TRAP protein regulates the ability of the tryptophan-activated TRAP protein to bind to trp operon leader RNA and promote transcription termination. AT synthesis is regulated both transcriptionally and translationally by uncharged tRNA(Trp). In this study, we examined the roles of AT synthesis and tRNA(Trp) charging in mediating physiological responses to tryptophan starvation.

Efficient expression of gene variants that harbour AGA codons next to the initiation codon.

In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons.

Ribosomal features essential for tna operon induction: tryptophan binding at the peptidyl transferase center.

Features of the amino acid sequence of the TnaC nascent peptide are recognized by the translating ribosome. Recognition leads to tryptophan binding within the translating ribosome, inhibiting the termination of tnaC translation and preventing Rho-dependent transcription termination in the tna operon leader region. It was previously shown that inserting an adenine residue at position 751 or introducing the U2609C change in 23S rRNA or introducing the K90W replacement in ribosomal protein L22 prevented tryptophan induction of tna operon expression.

Ribosome recycling factor and release factor 3 action promotes TnaC-peptidyl-tRNA Dropoff and relieves ribosome stalling during tryptophan induction of tna operon expression in Escherichia coli.

Upon tryptophan induction of tna operon expression in Escherichia coli, the leader peptidyl-tRNA, TnaC-tRNA(2)(Pro), resists cleavage, resulting in ribosome stalling at the tnaC stop codon. This stalled ribosome blocks Rho factor binding and action, preventing transcription termination in the tna operon's leader region. Plasmid-mediated overexpression of tnaC was previously shown to inhibit cell growth by reducing uncharged tRNA(2)(Pro) availability.

Excess of charged tRNALys maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature.

Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNA(Lys), of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41 degrees C whereas plasmids bearing other tRNA genes were ineffective. At 32 degrees C the excess of tRNA(Lys) did not alter the percentages of the free-, charged- or peptidyl-tRNA(Lys) species compared with those found in strains that did not overproduce tRNA(Lys).

Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide.

Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA(Pro), the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNA(Pro) peptide in facilitating tryptophan binding and induction.

Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.

Certain nascent peptide sequences, when within the ribosomal exit tunnel, can inhibit translation termination and/or peptide elongation. The 24 residue leader peptidyl-tRNA of the tna operon of E. coli, TnaC-tRNA(Pro), in the presence of excess tryptophan, resists cleavage at the tnaC stop codon.

Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons.

Minigenes encoding the peptide Met-Arg-Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the 'mini-ORFs' employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNA(Arg4), CGA is recognized by the abundant tRNA(Arg2).