Mechanism of translational initiation in prokaryotes. Evidence for a direct effect of IF2 on the activity of the 30 S ribosomal subunit.

Initiation factor IF2 from either Escherichia coli or Bacillus stearothermophilus was found to possess the previously undetected property of stimulating the template-dependent ribosomal binding of aminoacyl-tRNAs with free alpha-NH2 groups. IF1, which had no detectable activity alone, was found to stimulate the activity of E. coli IF2 and, to a lesser extent, that of B. stearothermophilus IF2. Since in the absence of ribosomes not even a weak interaction between the two IF2 molecules and the aminoacyl-tRNAs was detected, the present findings indicate that IF2 can act at the ribosomal level stimulating aminoacyl-tRNA binding without prior formation of a binary complex with the aminoacyl-tRNA. IF2 does not appear to open or strengthen a weak A-site binding, but rather to enhance aminoacyl-tRNA binding to a 30 S site equivalent to the P-site by slowing down the rate of aminoacyl-tRNA dissociation from ribosomes.

Relationship between size of mRNA ribosomal binding site and initiation factor function.

The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template. To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths. When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side. When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3. These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.

Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors.

The interaction between Escherichia coli 30S ribosomal subunits and mRNAs, and the effect of the initiation factors on this process, have been studied using MS2 RNA, polyribonucleotides and model mRNAs encoded by synthetic genes. The interactions were analyzed by gel filtration, by sucrose gradient centrifugation and by competition for ribosome binding between the various mRNAs and a Shine-Dalgarno deoxyoctanucleotide. It was found that the initiation factors do not significantly affect the Shine-Dalgarno interaction nor the apparent Ka values of the 30S-subunit-mRNA binary complexes, but influence the positioning of the mRNAs on the 30S subunit with respect to the Shine-Dalgarno octanucleotide. The results suggest that, in the absence of initiation factors, the mRNA occupies a ribosomal "stand-by" site which is close to or includes the region where the Shine-Dalgarno interaction takes place; in the presence of the factors, the mRNA is shifted away from the stand-by site, towards another ribosomal site with similar affinity for the mRNA. This shift does not require the presence of fMet-tRNA and, depending upon the type of mRNA, is mediated by IF-2 and/or IF-3.

Selection of the mRNA translation initiation region by Escherichia coli ribosomes.

Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka approximately 2 x 10(7) M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA.

Proteins from the prokaryotic nucleoid. A protein-protein cross-linking study on the quaternary structure of Escherichia coli DNA-binding protein NS (HU).

Escherichia coli DNA-binding proteins NS1, NS2 and NS (NS1 + NS2) react with the protein-protein bifunctional cross-linking reagents dimethylsuberimidate and dimethyladipimidate to yield oligomers up to hexamers. The former reagent, with the longer arm, is more efficient than the other shorter one. Both one- and two-dimensional gel electrophoreses show that the cross-linked trimers are homogeneous, while the dimers appear heterogeneous, suggesting that at least two types of dimers but geometrically equivalent trimers are formed. In the presence of DNA, the cross-linking reaction with either reagent yields fewer dimers and more of the larger products. The yield of cross-linked products of various sizes was determined for NS1, NS2 and NS as a function of the protein concentration (0.03-3000 microM). From the results obtained in these experiments, we derived a model of quaternary structure in which dimers and tetramers are predominant in very solutions of the proteins. Above a critical concentration (10-50 microM), interactions among tetramers become increasingly important, yielding octamers and perhaps larger products. Our data do not support a recently proposed model in which the DNA is packaged around a protein disc consisting of 8-10 NS dimers.