We propose that pyrrolid-2-one-5-carboxyl-tRNA is not involved in the initiation of protein synthesis in eukaryotic cells and that the N-terminal pyrrolid-2-one-5-carboxylic acid group of an IgG (immunoglobulin G) (that secreted by the mouse plasmacytoma Adj PC5) is formed by the enzymic cyclization of the N-terminal glutamine of the heavy chain of the completed IgG molecule and that the cyclization takes place inside the cell. We base these conclusions on the following evidence. (1) Pyrrolidonecarboxyl-tRNA was not found in incorporation experiments with rat liver preparations and [U-(14)C]-pyrrolidonecarboxylic acid, glutamic acid and glutamine, even though an incorporation extent of less than 2% of the total products could have been detected. (2) Double-labelling experiments showed that less than 8% of the nascent peptides of heavy chains (those obtained by precipitation by the antibody to Fc fragment) began with pyrrolidonecarboxylic acid. (3) Further double-labelling experiments showed that 60-66% of the heavy chains of the completed intracellular IgG molecule began with pyrrolidonecarboxylic acid after both 1 and 5h of labelling. (4) The IgG, after secretion by plasmacytoma Adj PC5, was found to have the sequence [unk]Glu- Val-Gln-Leu- at the N-termini of the heavy chains.
In rabbit reticulocytes iron deficiency causes depression of protein synthesis and a breakdown of polysomes. The effects of amino acid starvation are more complex. In all cases there is a depression of protein synthesis but only in some cases are changes in the size of the polysome observed. These findings prompted us to compare certain of the parameters that describe the synthesis of a-and ,8-chains of haemoglobin in normal and deprived reticulocytes. We have measured the relative numbers of ribosomes making each chain, the size of the polysomes involved in the synthesis of each chain, the distribution of the ribosomes on the aand ,B-chain messenger-RNA classes, and the rate of movement of ribosomes along the two different classes of messenger RNA.Our experiments show that under normal conditions, the rate oftranslation ofthe a-chain messenger RNA is approx. 1-3 times that of the fl-chain messenger RNA, and the polysomes synthesizing a-chains are smaller on average than those synthesizing fl-chains. Further, as far as can be measured, the distribution of ribosomes is uniform along both classes of messenger RNA. From these results we argue that the overall rate-limiting step in a-and fl-chain synthesis is not the terminal step of protein synthesis, but is probably the initiation process of chain assembly. Under special conditions, however, the rate-limiting step might be the assembly process itself. The rate of movement of ribosomes down the messenger RNA is not governed by a general step of protein synthesis, since it is affected by the sequence of nucleotides being translated. This control could operate through the availability of specific aminoacyl transfer RNA types, and starvation of particular amino acids can be shown to cause alteration in the balance of a-and fl-chain synthesis. For instance, starvation of threonine (which residue occurs 11 times in the a-chain and four times in the fl-chain) causes an increase in the time taken to translate the a-chain relative to the fl-chain. On the other hand our results and those of other workers suggest that haem or iron deficiency affects initiation of new chains and not the rate of movement of ribosomes as has previously been suggested.From the translation time and the number of ribosomes engaged in the synthesis of each chain it is possible to calculate the number of molecules of each chain made in unit time. Since there appear to be nearly equal numbers of ribosomes making a-and fl-chains, it follows that there is a continuous production of a small excess of a-chains relative to ,-chains. This surprising result is compatible with the existence of an a-chain pool in reticulocytes. These findings will be discussed in connection with the control of the assembly of haemoglobin in rabbit reticulocytes.
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