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Biomedical subjects

R Kaempfer

Publications and source records attributed to R Kaempfer.

70 records · Page 4Linked to original sources
PubMed1975

Initiation of protein synthesis. Binding of messenger RNA.

Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.

Bacterial Proteins↗
PubMed1975

Translational repression of a viral messenger RNA by a host protein.

It is shown that factor i, a bacterial protein, specifically inhibits that step in the initiation of R17 bacteriophage RNA translation that involves the attachment of native R17 RNA to 30 S ribosomal subunits carrying fMet-tRNA. This inhibition by factor i is relieved by the addition of excess R17 RNA, but not by the addition of excess 30 S subunits. That R17 RNA is the only target of the inhibition is demonstrated further by the fact that in a cell-free extract containing all components for protein synthesis, factor i-mediated inhibition of exogenous R17 RNA translation can be overcome only by the addition of excess R17 RNA and not by excess cell-free extract. Upon relief of inhibition, phage coat protein synthesis is restored; enhancement of formation of other cistron products is not seen. While initiation of R17 RNA translation is blocked by factor i, chain elongation is not affected. Although foactor i inhibits the IF-3-dependent binding of R17 RNA to fMet-tRNA-30 S complexes, under conditions of initiation of protein synthesis formation of stable complexes between factor i and IF-3 could not be detected, and factor i did not interfere with the binding of IF-3 to free, native R17 RNA. Instead of affecting the function of IF-3 or ribosomes, factor i exerts its inhibition by binding to R17 RNA and acting as a translational repressor. Factor i prefers intact R17 RNA to fragments generated by autoradiolysis; its binding to R17 RNA is specific in that little competition is observed by transfer RNA, ribosomal RNA or poly(A). However, factor i has a high affinity for poly(U) sequences.

Bacterial Proteins↗
PubMed1974

Role for heme in mammalian protein synthesis: activation of an initiation factor.

Two nonerythropoietic tissues, brian and liver, contain an initiation factor that can overcome the block in initiation of protein synthesis seen in reticulocyte lysates when exogenous hemin is not present. Upon incubation of the brain factor with hemin and removal of free hemin by gel filtration, the factor activity is strongly stimulated. This stimulation shows a concentration dependence on hemin close to that seen for stimulation of protein synthesis in whole reticulocyte lysates. The data indicate that hemin mediates the formation of an active initiation factor complex from inactive, lower molecular weight components.

Animals↗
PubMed1974

Sequence of events in initiation of translation: a role for initiator transfer RNA in the recognition of messenger RNA.

It is shown that initiation of translation involves several steps. (i) Binding of fMet-tRNA(fMet) to the bacterial 30S ribosomal subunit in the absence of messenger RNA, yielding a 34S complex. This binding is rapid and dependent on initiation factor 2 but not on initiation factor 3. (ii) Binding of messenger RNA to the 34S complex. This binding is slower and depends on initiation factor 3. If R17 RNA is used as messenger, the resulting complex sediments at 46 S. (iii) Joining of a 50S subunit to yield a complete initiation complex. Binding of fMet-tRNA(fMet) not only precedes, but is necessary for, correct binding of messenger RNA to ribosomes. Thus, initiator tRNA may play an active role in the selection of initiation sites in messenger RNA.

Anti-Bacterial Agents↗
PubMed1973

Inhibition of cellular protein synthesis by double-stranded RNA: inactivation of an initiation factor.

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA is caused by the inactivation of IF-3, an initiation factor required for the recycling of ribosomes and for their binding to messenger RNA. The evidence for this is that (i) the inhibition can be overcome by addition of exogenous If-3; (ii) double-stranded RNA inactivates stoichiometric amounts of IF-3; (iii) double-stranded RNA forms a complex with IF-3; and (iv) double-stranded DNA, which lacks inhibitory activity, also binds to IF-3, but with a much lower affinity than double-stranded RNA. It is concluded that double-stranded RNA inhibits cellular protein synthesis by tightly complexing with IF-3. It is suggested that IF-3 normally recognizes a double-stranded region in messenger RNA.

Amino Acids↗
PubMed1972

Translational control of hemoglobin synthesis by an initiation factor required for recycling of ribosomes and for their binding to messenger RNA.

The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 37 degrees requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes.These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor.

Animals↗
PubMed1971

Control of single ribosome formation by an initiation factor for protein synthesis.

30 and 50S ribosomal subunits, released from polysomes upon polypeptide chain termination, possess a high affinity for each other and readily form single ribosomes. Highly purified initiation factor F3(B), acting stoichiometrically, prevents the formation of single ribosomes without promoting their dissociation. These findings are interpreted in terms of a ribosome cycle in which a limiting amount of factor F3(B) controls the number of ribosomes active in protein synthesis, in response to metabolic changes in the cell, by regulating the flow of ribosomal subunits into polysomes or into a sidetrack pool of synthetically inactive single ribosomes. The reported apparent ribosome dissociation activity of F3(B) is explained.

Anti-Bacterial Agents↗