Ribosomes stalled

Ribosome stalls at the his codons and prevents terminator formation... 

When tryptophan concentrations are high, concentrations of charged tryptophan tRNA (Trp-tRNATrp) are also high. This allows translation to proceed rapidly past the two Trp codons of sequence 1 and into sequence 2, before sequence 3 is synthesized by RNA polymerase. In this situation, sequence 2 is covered by the ribosome and unavailable for pairing to sequence 3 when sequence 3 is synthesized the attenuator structure (sequences 3 and 4) forms and transcription halts (Fig. 28-21b, top). When tryptophan concentrations are low, however, the ribosome stalls at the two Trp codons in sequence 1, because charged tRNATrp is less available. Sequence 2 remains free while sequence 3 is synthesized, allowing these two sequences to base-pair and permitting transcription to proceed (Fig. 28-21b, bottom). In this way, the proportion of transcripts that are attenuated declines as tryptophan concentration declines. 

Figure 29-16 Schematic diagram of the tmRNA structure and its function in the rescue of ribosomes stalled at the end of a messenger RNA that has been broken and has lost its inframe termination codon. After it binds into the ribosomal A site the tmRNA, which has been charged with alanine, undergoes the peptidyltransferase reaction and translocation to the P site. Then it lays down its mRNA-like coding sequence, which is used by the ribosome to add ten more amino acids to form the 11-residue C-terminal degradation signal A ANDENYALAA. This induces rapid degradation of the imperfect protein that has been formed.4363...

Fig. 2. Attenuation of the trp operon. (a) When tryptophan is plentiful, sequences 3 and 4 base-pair to form a 3 4 structure that stops transcription (b) when tryptophan is in short supply, the ribosome stalls at the trp codons in sequence 1, leaving sequence 2 available to interact with sequence 3. Thus a 3 4 transcription terminator structure cannot form and transcription continues.

Fig. 5. Protein-RNA fusion. Covalent RNA-protein complexes can be generated by ligation of a DNA-puromycin linker to the in vitro transcribed mRNA. During in vitro translation, the ribosome stalls at the RNA-DNA junction. Puromycin can then bind to the ribosomal A-site. The nascent polypeptide is thereby transferred to puromycin. The resulting covalendy linked complex of mRNA, puromycin, and peptide can be used for selection experiments. After affinity selection, the bound complexes are eluted and subsequently the mRNA is amplified by RT-PCR.

The trp operon has no positive control system like cAMP-CAP, but it does have an additional transcriptional control mechanism that depends on the concentration of tryptophan. This involves the attenuator site, which resides within the leader (L) sequence. It consists of 14 adjacent codons beginning with a methionine codon (AUG) and ending with a termination codon (UGA) and, importantly, codons (UGG) for tryptophan at positions 10 and 11. When tryptophan is plentiful, the complete 14-residue polypeptide (leader polypeptide) is synthesized. When tryptophan is scarce, the ribosome stalls at the tandem UGG... 

The mechanism of ribosome stalling during translation of tnaC has been investigated in considerable detail. The nascent leader peptide (TnaC) interacts with ribosomal protein L22 and 23 S rRNA in the narrow region of the ribosome exit channel (14). Ribosome recognition of the TnaC peptide results in specific binding of free tryptophan in the ribosome, which inhibits cleavage and the subsequent release of the nascent leader peptide (15, 16). 

Gong M, Cruz-Vera LR, Yanofsky C. Ribosome recycling factor and release factor 3 action promotes TnaC-pep tidyl-tRNA dropoff and relieves ribosome stalling during tryptophan induction of tna operon expression in Escherichia coli. J. Bacteriol. 2007 189 3147-3155. 

Gene expression can also be regulated at the level of translation. In prokaryotes, many operons important in amino acid biosynthesis are regulated by attenuation, a process that depends on the formation of alternative structures in mRNA, one of which favors transcriptional termination. Attenuation is mediated by the translation of a leader region of mRNA. A ribosome stalled by the absence of an aminoacyl-tRNA needed to translate the leader mRNA alters the structure of mRNA so that RNA polymerase transcribes the operon beyond the attenuator site. 

Tsai CJ, Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM, Nussinov R (2008) Synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima. J Mol Biol 383 281-291... 

Figure 4 Puromycin (A) is an antibiotic analog of tyrosyl tRNAthat differs by the groups in red. Once joined to a DNA linker at the 3 -end and a psoralen (B) at the 5 -end it is ready to covalently link the mRNA to nascent peptide. Photoactivation of the psoralen (green), cross-links the 5 -end of the linker and the mRNA. Once the ribosome stalls at the end of the mRNA, the puramycin enters the ribosome A-site and is transferred to the end of the newly formed protein. However, the ribosome is unable to hydrolyze the ribamine (red) amide bond thus forming a permanent link between mRNA and the encoded protein (C).

Figure 5 Starting from natural mRNA, a cDNA library (A blue) is produced and like ribosomal display, the cDNA is transcribed into mRNA (B) with no stop codons. The 3 -end of each mRNA molecule is ligated to a short synthetic DNA linker (C) and sometimes a polyethyleneglycol spacer, which terminates with a puramycin molecule (small red sphere). The ligation is stabilized by the addition of psoralen (green clamp), which is photoactivated to covalently join both strands. Addition of crude polysomes or purified ribosomes (D) results in translation of the mRNA into protein, but the ribosome stalls at the mRNA-DNA junction. Since there are no stop codons, release factors cannot function and instead the puromycin enters the A-site of the ribosome (A). Because puramycin is an analog of tyrosyl-tRNA, the peptidyl transferase subunit catalyzes amide bond formation between the puromycin amine and the peptide carboxyl terminus, but is unable to hydrolyze the amide link (which should be an ester in tyrosyl-tRNA) to release the dimethyladenosine. The ribosome is dissociated to release the mRNA-protein fusion (E), which is protected with complementary cDNA using RT-PCR (F). The mRNA library can then be selected against an immobilized natural product probe (G), nonbinding library members washed away and the bound mRNA (H) released with SDS. PCR amplification of the cDNA provides a sublibrary (A) for another round of selection or for analysis/ sequencing.

Figure 16 The model for the mechanism of macrolide-dependent ribosome stalling proposed by Vazquez-Laslop ef al.

Fig. 16.8. Attenuation of the trp operon. Sequences 2, 3, and 4 in the mRNA transcript can form base pairs (2 with 3 or 3 with 4) that generate hairpin loops. When tryptophan levels are low, the ribosome stalls at the adjacent trp codons in sequence 1, the 2-3 loop forms, and transcription continues. When tryptophan levels are high, translation is rapid and the ribosome blocks formation of the 2-3 loop. Under these conditions, the 3 loop forms and terminates transcription.

If tryptophan is limiting, the ribosome stalls out over the tryptophan codons on the mRNA of the leader sequence. This leaves the mRNA free to form the... 

FIGURE 11.16 The attenuation mechanism in the trp operon. The pause structure forms when the ribosome passes over the Trp codons quickly when tryptophan levels are high. This causes premature abortion of the transcript as the terminator loop is allowed to form. When tryptophan is low, the ribosome stalls at the Trp codons, allowing the antiterminator loop to form, and transcription continues. 

R 42 P.W. Haebel, S. Gutmann and N. Ban, Dial tm for Rescue tmRNA Engages Ribosomes Stalled on Defective mRNAs , p. 58... 

Secondary structures of eukaryotic mRNAs also critically influence the initiation and termination of protein synthesis (153). For instance, a strong stem-loop structure (AG = —30 kcal/mol) prevents a mRNA from engaging the 40S ribosomal subunit when the secondary structure occurs at 12 nt from the cap. Another stem—loop structure with a AG of —61 kcal/mol results in ribosome stalling, probably because the structure is too stable to be unwound by the 40S subunit. 

Ude S, Lassak J, Starosta AL et al (2013) Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science 339 82-85... 

---