Tetranucleotides as Effectors for the Binding of Initiator tRNA to Escherichia coli Ribosomes

Abstract

Oligonucleotides such as G-A-G-G, which are complementary to the C-U-C-C region at the 3' end of 16-S RNA, inhibit the R17-RNA-dependent binding of the initiator tRNA (fMet-rRNA) to 30-S ribosomal subunits. However, if phage RNA is replaced by A-U-G, the same oligonucleotides stimulate the binding of fMet-tRNA to the 30-S subunits. This indicates that the formation of the RNA x RNA hybrid acts as a positive control signal for the selection of the initiator tRNA by the 30-S-subunit x mRNA complex. Tetranucleotides of the type A-U-G-N (where N = A, G, C or U) stimulated the IF-2-dependent binding of fMet-tRNA to the 30-S subunit more effectively than A-U-G, with A-U-G-R better than A-U-G-Y (where R is a purine nucleoside and Y is a pyrimidine nucleoside). Since the 3'-terminal adenosine in A-U-G-A can be replaced by 6-deamino-adenosine, a stacking type of interaction between U-33 of tRNA and N of A-U-G-N should additionally stabilize the codon-anticodon complex. The situation is strictly reversed for 70-S ribosomes where A-U-G is the best codon followed by A-U-G-U, A-U-G-C, A-U-G-G and A-U-G-A. Replacement of GTP by guanosine 5'-[beta, gamma-methylene]triphosphate (GuoPP[CH2]P] results in A-U-G-A becoming more efficient than A-U-G as the codon for the binding of fMet-tRNA to 70-S ribosomes. This indicates that IF-2 and GTP hold the anticodon of the fMet-tRNA in a conformation capable of binding to a tetranucleotide codon. GTP hydrolysis and release of IF-2 from the 70-S ribosome results in a change of the tertiary structure of fMet-tRNA as a consequence of which the initiator tRNA reassumes the conformation which preferentially binds to A-U-G.

A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

“…Analysis of our results has also provided a more complete picture of the ribosome-ribosome interactions that are responsible for ribosome re-initiation. First, the re-initiation rate is substantially higher ( k reinitiation = 0.022) at d = −4 because the 30S ribosomal complex's tRNA fMet anti-codon loop (3′-UACU-5′) forms four Watson–Crick base pairings with the intergenic sequence (5′-AUGA-3′) ( 47 ) and scanning is not required to re-initiate translation. Second, when scanning is necessary for ribosome re-initiation, the rate of translation appears to be governed by a one-dimensional random walk along the mRNA with spontaneous detachment.…”

Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.

“…(a) Ribosome binding studies carried out with synthetic AUG-containing oligonucleotides suggest involvement of the nucleotide immediately preceding the initiator codon (position -1) and the nucleotide immediately following the initiator codon (position +4). A pyrimidine in position -1 promotes the highest level of oligonucleotide binding (102,125), whereas a purine is most effective in position +4 (403,404). (The latter effect was not observed in all experiments, however [125].)…”

The Context Theory as Applied to the Decoding of the Initiator tRNA by Escherichia coli Ribosomes

“…The latter investigators have also presented evidence that the 5'-uridine in U-A-U-G strengthens the binding of the initiator tRNA to the 30-S subunit. We have already documented that A-U-G-R-type tetranucleotides are more efficient initiation signals than A-U-G-Y or A-U-G [6,7]. Further support for the concept of a four-nucleotide codon-anticodon complex comes from the unusual anticodon loop structure of tRNA:"' [8] : uridine-33 occupies an exposed position and adenosine-37 is unmodified, features not found in elongator tRNAs [9,10].…”

“…Initiation factors were obtained as described [18], except that in an additional step IF-2 was concentrated by adsorption to phosphocellulose. IF-1 and IF-2 were not separated from each other Oligonucleotides were synthesized with polynucleotide phosphorylase as described in [7,17]. Their homogeneity was examined by high-pressure liquid chromatography (HPLC) (Zorbax@-NH2, DuPont Instruments LC SSO), and their nucleoside composition was verified with the aid of a nucleoside analyzer [19].…”

“…Prior to use they were activated as described by Zamir et al [16]. Ribosomes and subunits were characterized for their biochemical functions as reported in [7,17]. Initiation factors were obtained as described [18], except that in an additional step IF-2 was concentrated by adsorption to phosphocellulose.…”