Rob Benne

INITIATION OF EUKARYOTIC PROTEIN SYNTHESIS

The initiation of protein synthesis is defined as the sequence of events, which leads to an 80 S . MettRNA . mRNA complex. Several non-ribosomal proteins are required for the formation of this 80 S initiation complex and they are called eIF-1, -2, etc. (eukaryotic initiation factor). They are listed in table 1, with the effect they have on protein synthesis and on partial reactions thereof, as desiribed below. An initiation factor is defined as a protein which stimulates one or more of these, and only these reactions, and which is released after the completion of an 80 S initiation complex, in contrast with a ribosomal protein, which remains an integral part of the ribosome during all stages of protein synthesis. Dissociation of 80 S ribosomes is a prerequisite for the initiation of eukaryotic protein synthesis, since the initial binding of Met-tRNA occurs on a 40 S subunit and not on an 80 S ribosome. Spontaneous

THE ROLE OF eIF-4C IN PROTEIN SYNTHESIS INITIATION COMPLEX FORMATION

eIF-4C has a pronounced stimulatory effect on initiation complex formation with native 80-S ribosomes (80-Sn) as the only source of ribosomal subunits, but only a small effect when washed 40-S subunits are used. eIF-4C is accessary to eIF-3 in dissociating 80-Sn ribosomes. eIF-4C is present on 40-Sn but absent on 40-Sn dimers, which occur in preparations of native ribosomes and are as such inactive in protein synthesis. eIF-4C dissociates 40-Sn dimers into active monomers. These results can be explained by assuming that the presence of eIF-4C on 40-Sn prevents: (a) premature association with 60-S ribosomal subunits and (b) dimerisation, thus increasing the rate and extent of initiation complex formation.

Initiation of protein synthesis in neuroblastoma cells infected by Semliki forest virus A decreased requirement of late viral mRNA for eIF-4B and cap binding protein

Semliki Forest Virus causes shut-off of protein synthesis in its mammalian hosts [ 1,2]. Usually, this shutoff takes place 3-5 h post-infection [2]. In neuroblastoma cells from mouse such an event leads to the exclusive production of late viral (= structural) proteins (e.g., ~97, ~62, El and capsid protein, see [2--41). These proteins are synthehized as a large precursor from a sub-genomic template of 26 S [ 1 ,.S] an mRNA which appears late in infection and is identical to the 3’-terminal part (-1/3rd) of the viral genomic RNA of 42 S [6,7]. This 42 S mRNA serves as a template early in infection for the synthesis of non-structural proteins [2,4]. The genes for these proteins are located at the 5’-terminus of this messenger, whereas the genes for the structural proteins, which are found at the 3’-part of 42 S are silent (see above, [2]). At a late stage of infection, 26 S mRNA is the only messenger recognized by the protein synthesizing machinery, despite of the fact that still considerable amounts of 42 S and host mRNAs are present in the infected cell [1,%31. High salt washes of ribosomes (containing the initiation factors of protein synthesis), isolated from neuroblastoma cells at a late stage of infection by SFV, have mostly lost the ability to support the trsnslation in ‘in vitro’ systems of hostand early viral mRNA, although they are still highly active with viral lateand EMC mRNA [9]. Addition to systems programmed with hostand viral early mRNA, of purified initiation factors eIF4B (M, 80 000, [9,10]) and cap binding protein (CBP, MI 24 000, [9,1 l]), but none of the others significantly increases the activity of these infectionexposed crude initiation factors. These fmdings indicate that the lesion in the protein synthesizing machinery has occurred at the level of initiation factors eIF4B and CBP, although the precise mechanism by which these factors are incapacitated remains, at present, unclear. Here we substantiate the ability of purified eIF4B and CBP to stimulate the activity of infectionexposed crude factors in protein synthesis in an ‘in vitro’ system programmed with early viral mRNA. We explore why the SFV-induced blockade of eIF4B and CBP activity results in a preferential recognition of late SFV mRNA by the protein synthesizing machinery in infected cells. We show that optimal translation of 26 S mRNA requires a 2-4-times smaller amount of eIF4B and CBP when compared to hostand 42 S mRNA. Such a low requirement for eIF4B and CBP results in a relatively undisturbed translation of viral late mRNA in infected cells in spite of the fact that these factors are gradually inactivated. The reason for the decreased need for eIF4B and CBP, factors which are thought to be involved in the recognition of the 5’-terminal cap structure of mRNAs [ 12-141 is not based on a decreased importance of the cap-structure of 26 S mRNA, since chemically decapped 26 S RNA almost completely looses its capacity to serve as a template in pH 5 systems.

Mode of action of protein synthesis initiation factor eIF-1 from rabbit reticulocytes

Initiation of eukaryotic protein synthesis is mediated nby a set of protein factors, purified using predominantly nrabbit reticulocytes and Krebs II nascites cells. At least 8 initiation factors with Mr n15000-700000 could be described. This elaborate nwork was followed by the functional characterization nof the individual factors which provided considerable ninsight in the specific contribution of each factor nduring the assembly of an initiation complex, nreviewed.

In vitro translation of semliki forest virus 42 S RNA Initiation at two different sites

The genome of α-viruses like Semliki Forest virus n(SFV) and Sindbis virus consists of a single-stranded nRNA molecule of Mr 4-4.5 X l0⁶. The genes ncoding for the non-structural proteins are located near nthe 5 (capped) terminus of this 42 S RNA, whereas nthe information for the structural proteins, one capsid nand three envelope proteins, is found near the 3’ nterminus.

The Control of the Rate of Initiation of Eukaryotic Protein Synthesis

Publisher Summary This chapter discusses the control of the rate of initiation of eukaryotic protein synthesis. Control of the rate of initiation of protein synthesis is manifested at many different occasions during the lifetime of an average eukaryotic cell. During the cell cycle, there is no initiation of protein synthesis in mitosis. In the late S phase and in G2 phase, a gradual diminishing of initiation occurs. Under physiological stress—such as serum deprivation, amino-acid starvation, and hyperthermic treatment—the initiation rate is lowered substantially. The model for the control of the rate of initiation implies that when a complex is formed between eIF-2 and eRF, a high rate of initiation is achieved, whereas conditions that impair complex formation inevitably cause a low initiation rate. Reduced eIF-2 displays a much higher activity in lysate systems than oxidized eIF-2.

Purification and characterization of protein synthesis initiation factor eIF-3 from wheat germ

eIF-3 from wheat germ is a large multicomponent factor. It sediments at 15 S and is comprised of ten different polypeptides with an Mr value ranging from 26 000 to 135 000; five out of the ten seem to be present in a 1 : 1 stoichiometric ratio, whereas the others appear to occur approximately in a 0.5 to 1 ratio. The factor is active in a partially purified cell-free system derived from wheat germ and in a mammalian model assay system for the synthesis of methionyl puromycin.

The ATP-dependent DNAase from Escherichia coli rorA: A nuclease with changed enzymatic properties

The ATP-dependent DNAases from Escherichia coli wild-type and rorA were isolated and purified and their enzymatic properties were compared. The enzymes were found to differ in the amount of ATP that is consumed during DNA degradation. This difference can be influenced by the reaction conditions and the nature of the substrate.