Erythropoietin EPO Signaling Pathways Discussion

A clever experiment produced excellent evidence for the importance of base paring, not a particular sequence, between the Shine-Dalgarno sequence and the 3’-end of the 16S rRNA. Describe the experiment.

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(the next paragraph needs to paraphrase)

Anna Hui and Herman De Boer produced excellent evidence for the importance of base pairing between the Shine–Dalgarno sequence and the 39-end of the 16S rRNA in 1987. They cloned a mutant human growth hormone gene into an E. coli expression vector bearing a wild-type Shine–Dalgarno (SD) sequence (GGAGG), which is complementary to the wild-type 16S rRNA anti-SD sequence (CCUCC). This gave high levels of human growth hormone protein. Then they mutated the SD sequence to either CCUCC or GUGUG, which would not base-pair with the anti-SD sequence on the 16S rRNA. Neither of these constructs produced very much human growth hormone. But the clincher came when they mutated the anti-SD sequence in a 16S rRNA gene (on the same vector) to either GGAGG or CACAC, which restored the base pairing with CCUCC and GUGUG, respectively. Now the mRNA with the mutant CCUCC SD sequence was translated very well by the mutant cells with the 16S rRNA having the GGAGG anti-SD sequence, and the mRNA with the mutant GUGUG SD sequence was translated very well in cells with the 16S rRNA having the CACAC anti-SD sequence. This kind of intergenic suppression is strong evidence that important base-pairing occurs between these sequences.

How did the scientists figure out that there are three sites on ribosome (P, A, and E sites). Explain.

The answer is these paragrapgs : please summary it and paraphrase it.

The link between puromycin and the two-site model is this: Before translocation, because the A site is occupied by a peptidyl-tRNA, puromycin cannot bind and release the peptide; after translocation, the peptidyl-tRNA has moved to the P site, and the A site is open. At this point puromycin can bind and release the peptide. We therefore see two states the ribosome can assume: puromycin reactive and puromycin unreactive. Those two states require at least two binding sites on the ribosome for the peptidyl-tRNA. Puromycin can be used to show whether an aminoacyltRNA is in the A or the P site. If it is in the P site, it can form a peptide bond with puromycin and be released. However, if it is in the A site, it prevents puromycin from binding to the ribosome and is not released.

This same procedure can be used to show that fMettRNA goes to the P site in the 70s initiation complex. In our discussion of initiation in Chapter 17, we assumed that the

fMet-tRNAf Met goes to the P site. This certainly makes

sense, because it would leave the A site open for the second aminoacyl-tRNA. Using the puromycin assay, M.S. Bretscher and Marcker showed in 1966 that it does indeed go to the P site. They mixed [35S]fMet-tRNAf Met with ribosomes, the trinucleotide AUG, and puromycin. If AUG attracted fMet-tRNAf Met to the P site, then the labeled fMet should have been able to react with puromycin, releasing labeled fMet-puromycin. On the other hand, if the fMettRNAf Met went to the A site, puromycin should not have been able to bind, so no release of labeled amino acid

should have occurred. Figure 18.12 shows that the fMet attached to tRNAf Met was indeed released by puromycin,

whereas the methionine attached to tRNAm Met was not. Thus, fMet-tRNAf Met goes to the P site, but methionyl-tRNAm Met goes to the A site. One could argue that it was the fMet, not the tRNAf Met that made the difference in this experiment. To eliminate that possibility, Bretscher and Marcker performed the same experiment with Met-tRNAf

Met and found that its methionine was also released by puromycin (Figure 18.12c). Thus, the tRNA, not the formyl group on the methionine, is what targets the aminoacyl-tRNA to the P site.

Actually, x-ray crystallography studies in 2009 showed

that fMet-tRNAf Met does not automatically go to the P site. Instead, on its own, it goes fi rst into a hybrid state called the P/I state in which the anticodon of the tRNA is in the P site of the 30S subunit, but the fMet and acceptor stem of the tRNA are not in the P site of the 50S subunit, which encompasses the peptidyl transferase center. Instead, the fMet and acceptor stem are in an “initiator” site to the left of the P site (toward the E site) as the ribosome is conventionally depicted. It is the job of a protein factor called EF-P to bind to the left of fMet-tRNAf

Met and nudge the fMet and acceptor stem to the right into the peptidyl transferase center. That action puts the fMet-tRNAf Met fully in the P site.

In 1981, Knud Nierhaus and coworkers presented evidence

for a third ribosomal site called the E site. Their experimental strategy was to bind radioactive deacylated

tRNAPhe (tRNAPhe lacking phenylalanine), or Phe-tRNAPhe, or acetyl-Phe-tRNAPhe to E. coli ribosomes and to measure the number of molecules bound per 70S ribosome. Table 18.2 shows the results of binding experiments carried out in the presence or absence of poly(U) mRNA. Only one molecule of acetyl-Phe-tRNAPhe could bind at a time to a ribosome, and the binding site could be either the A site or P site. On the other hand, two molecules of Phe-tRNAPhe could bind, one to the A site, and the other to the P site. Finally, three molecules of deacylated tRNAPhe could bind. We can explain these results most easily by postulating a third site that presumably binds deacylated tRNA on its way out of the ribosome. Hence the E, for exit. In the absence of mRNA, only one tRNA can bind. This can be either deacylated tRNAPhe or acetyl-Phe-tRNAPhe. Nierhaus and colleagues speculated that the binding site was the P site, and subsequent work has confi rmed this suspicion.