Raymond Kaempfer

Cyclic dissociation into stable subunits and re-formation of ribosomes during bacterial growth

Abstract Studies of the distribution of isotopic labels among ribosomes and ribosomal subunits following transfer of a growing bacterial culture from a heavy- to a light-isotopes medium show that: (1) During growth ribosomes frequently undergo subunit exchange, presumably by dissociation into their 30 s and 50 s subunits and re-formation from a pool of free subunits. (2) Bibosomal subunits are stable: they remain intact during growth and are continuously, recycled through ribosomes. On the basis of these results, it is suggested that ribosomes must dissociate into their subunits between successive rounds of translation.

Identification and RNA-binding properties of an initiation factor capable of relieving translational inhibition induced by heme deprivation or double-stranded RNA

An initiation factor from rabbit reticulocytes can overcome the block in initiation of protein synthesis occurring in reticulocyte lysates when exogenous hemin is not present, or when double-stranded RNA is added. This factor has been identified with IF-MP, an initiation factor capable of forming ternary complexes with GTP and methionyl-tRNAF. Initiation factor IF-M3 by itself is unable to overcome the block in initiation, but appears to stimulate this activity of IF-MP. IF-MP binds to single-stranded R17 RNA as well as to double-stranded RNA, while IF-M3 only binds to double-stranded RNA. The protein synthetic activity of IF-MP is sensitive to N-ethylmaleimide, but its ability to bind RNA is resistant.

Initiation factor IF-3: a specific inhibitor of ribosomal subunit association

Abstract At Mg 2+ concentrations optimal for protein synthesis on natural messenger UNA, initiation factor IF-3 † effectively inhibits the association of 30 s and 50 s ribosomal subunits but does not cause the dissociation of single ribosomes. The rate of exchange between single ribosomes and ribosomal subunits is very slow at these concentrations of Mg 2+ , but increases markedly at lesser concentrations of Mg 2+ . The net dissociation of single ribosomes observed at low concentrations of Mg 2+ in the presence of IF-3 is not stimulated by this factor, but proceeds only to the extent of spontaneous dissociation of single ribosomes, with IF-3 subsequently intercepting the ribosomal subunits. The inhibition of ribosomal subunit association is displayed by highly purified IF-3, but not by IF-1 or IF-2. It can be overcome in the presence of 0.01 mspermidine, but not by putrescine. Ribosomal subunit association also is inhibited by tetracycline; this inhibition likewise is overcome by spermidine.

Host interference with viral gene expression: Mode of action of bacterial factor i

The mechanism of interference with R17 viral RNA expression by a host protein, factor i, was studied. Formation of initiation complexes on native bacteriophage R17 RNA molecules, as well as translation of R17 RNA in vitro, is blocked almost quantitatively by factor i. This inhibition is readily overcome by the addition of excess R17 RNA. Extensive complex formation between factor i and R17 RNA occurs during inhibition of initiation complex formation. Moreover, the extent of inhibition of R17 RNA translation correlates closely with the extent of complex formation between factor i and R17 RNA, and exhibits the same sigmoid concentration dependence on factor i. Although initiation complex formation is totally dependent upon initiation factor IF-3, neither this function of IF-3, nor its ability to prevent the association of 30 S and 50 S ribosomal subunits into single ribosomes, is affected by factor i. IF-3, even when present in tenfold molar excess over factor i, fails to relieve the inhibition of initiation on R17 RNA. It is concluded that factor i is a translational represser acting directly on messenger RNA. It is suggested that this repression is cistron-specific, affecting only viral coat protein synthesis. Messenger RNA discrimination by factor i does not involve initiation factor IF-3.

Resistance of bacterial protein synthesis to double-stranded RNA☆

Abstract Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.

[56] Sedimentation velocity analysis in accelerating gradients

Publisher Summary Zone sedimentation is extensively employed for the separation and analysis of macromolecules and macromolecular complexes, and to obtain stably sedimenting bands, preformed density gradients are usually used as supporting media. This chapter describes various gradients for better separation in which the viscosity decreases with increasing concentration. In these accelerating gradients, the combination of increasing centrifugal force and decreasing viscous drag allows a substantial increase in sedimentation velocity as a function of distance sedimented. Sucrose and glycerol are the solutes most commonly chosen for the construction of such gradients. The faster sedimenting particle is accelerated over the slower sedimenting one, leading to a separation greater than proportional to the difference in their sedimentation coefficients. The accelerating gradient is most useful in separating particles possessing closely similar sedimentation coefficients.

Masking of peptidyl transferase activity in polyribosomes.

Abstract The peptidyl transferase activity of polysomes from Escherichia coli , rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.

[48] Ribosomal subunit exchange: Analysis in vivo

Publisher Summary This chapter describes the subunit exchange in ribosomes that can be studied in vivo by centrifugal analysis of the distribution of isotopic labels among ribosomes and ribosomal subunits following transfer of cells uniformly labeled with heavy isotopes to a medium containing only light isotopes. The technique is based on the fact that at the time of transfer, the cell contains only heavy ribosomes and ribosomal subunits. During growth in light medium exchange of subunits among ribosomes is manifested by the progressive replacement of the originally heavy ribosomes by two species of hybrid density. In Escherichia coli, one of these two hybrid species contains a heavy 50 S and a light 30 S subunit, the other a light 50 S and a heavy 30 S subunit. Thus, formation of ribosomes of hybrid density may be detected by a variety of ultracentrifugal methods. The density assay for ribosomal subunit exchange can be used with all organisms capable of growing on heavy isotopes, and this technique allows a direct demonstration of the cyclic dissociation into subunits and reformation of ribosomes under conditions of normal growth.