B.F.C. Clark

The role of N-formyl-methionyl-sRNA in protein biosynthesis

Bacterial sRNA has been fractionated into two methionine-accepting species, only one of which could be formylated. The binding of each charged species to ribosomes under the direction of a variety of trinucleoside diphosphates has been studied. The codeword AUG could be assigned to both species, whereas the related trinucleoside diphosphates GUG and UUG caused binding only of the species which could be formylated. The presence of the formyl group on this methionyl-sRNA did not alter its binding characteristics. The results of the binding studies were supported by investigations using a cell-free system directed by poly UAG or poly UG. Methionine was incorporated from the charged species, which could be formylated into the N-terminal position of polypeptides by both polymers whether this species was formylated or not. Poly UAG, but not poly UG, stimulated the incorporation of methionine into polypeptide from the methionyl-sRNA species, which could not be formylated. The analysis of this polypeptide product identified methionine in internal positions.

High-resolution X-ray diffraction studies on a pure species of transfer RNA

Systematic crystallization studies on yeast transfer RNAphe have yielded crystals large enough for X-ray crystallography. The crystal form is monoclinic; P21 a = 56.0 A, b = 33.4 A, c = 63.0 A and β = 90.4 °. The X-ray diffraction patterns extend to beyond 3 A spacings.

Stability of different ribosomal complexes with initiator transfer RNA and synthetic messenger RNA

Abstract Stability of the mRNA-initiator tRNA-ribosomal complex has been studied by nitrocellulose filter binding and thermal dissociation techniques. There is no significant difference between the stability of the 70 s ribosomal complex involving fMet-tRNA f § and its coding triplet and the stability of other synthetic mRNA-aminoacyl-tRNA systems including the ApUpG-Met-tRNA f complex. However, the 30 s subunit complex with fMet-tRNA f and ApUpG is significantly more stable than Met-tRNA f -ApUpG and other aminoacyl-tRNA-mRNA complexes. Further, the 30 s subunit complex with acetylphenylalanyl-tRNA and poly U is unstable. A stable 70 s ribosomal complex with Met-tRNA m is formed with poly (A,U,G) but not with the triplet ApUpG.

Formylation of mischarged E. coli tRNA Met f

ha previous work we demonstrated the mischarging of fG&erichio coli tRNAF” by yeast phenyManyC and va&CtRNA synthetam [ 1 . The possibility of kI obtaining phenylalanyl-tRNA, ct and valy CtFWA~” allows the study of &he properties of these rnisdlarged +&of initiator t&WA durlag the different steps of ibe idtiatidn process of protehb synthesis. T&e first reaction of the initiation mechanism in JF. cofi is thf~ formy’_tton of methionyl-tRNAF”. It was therefore _=q flr3t to ve.rkfy if an incorrectly aminoacylated initiitc

Inhibition of translation initiation complex formation by MS1

!RNA is able to be formylated. In the present paper we demonstrate that eilher ph*oylalsnyl-tRNA~” orvalyl-tRNAys . cafl be formylaced in the presenw of the tkmsfonnylase from & coli. Thcsz results suggst that the specifkity of the formylation reaction exclusively depends upon the nature of the tRNA moiety of the aminoacylated tRNAF”’ and not upon tit of the amG10 acid bound to the tRNA.

THE NUCLEOTIDE SEQUENCES OF CYTOPLASMIC METHIONINE AND VALINE tRNAS FROM MOUSE MYELOMA CELLS

When stringent cells of Escherichi coli are starved for an amino acid, a guanine nucleotide derivative, MS1 , identified as ppGpp [l] , rapidly accumulates to relative high concentrations [2, 31 . The level of this derivative usually exceeds that of guanosine triphosphate [3,4], GTP (pppG), which in turn is in excess over guanosine diphosphate, GDP (ppG) [5]. In general, the level of MS1 can be directly correlated with the rate of cell growth [6] . We show here that MS1 can inhibit a step in protein synthesis; specifically the formation of the initiation complex containing formylmethionyltRNAf, 70 S ribosomes and messenger RNA.

Location of a platinum binding site in the structure of yeast phenylalanine transfer RNA

The primary structure of the cytoplasmic initiator tRNA (tRNAf Me*) of mammalian cells has recently been determined [l-4] . This tRNA has a structure specially adapted for its role of providing the N-terming methionyl residue of newly synthesised polypeptides. tRNApet is also discriminated against during the elongation phase of protein synthesis since Met-t~A~et has no appreciable ability to donate methionine for polypeptide chain elongation. For comparison purposes it would be interesting to determine the primary structure of a tRNAMe* which functions in protein elongation in mammalian cells, but which is totally inoperative as an initiator tRNA. We report here the nucleotide sequence of one such mammalian tRNAMet species. This tRNA was purified from mouse myeloma cells and it corresponds to the species which has been designated tRNATet [5,6] on the profile of the RPC-5 column chromatoi graphic separation of the isoaccepting methionine tRNAs of mammalian cells, In addition we also report the primary structure of the major valine tRNA of mouse myeloma cells, since the structure of this tRNA was found unexpectedly to throw some light upon present knowledge concerning the structure-function relationship of the mammalian methionine tRNAs. Details of the sequencing of mouse myeloma tRNApe* and tRNAVal will be published elsewhere. As well as the nucleotide sequences discussed in this paper, the sequences of two other mammalian tRNAs, a rat liver tRNASer [7] and a rabbit liver tRNAPhe [8] have previously been reported. 2. Experimental

Lack of Tpψp in loop IV of a mammalian initiator transfer RNA

Abstract Trans-dichlorodiammineplatinum (II) reacts with yeast phenylalanine transfer RNA to yield a major platinum binding site. The tightly bound platinum has been located on the oligonucleotide Gm-A-A-Y-A-ψp containing the anticodon by standard fingerprinting methods using 32P-labelled tRNAPhe. This site corresponds to a single major platinum site identified during an X-ray crystallographic analysis of yeast tRNAPhe. The solution studies have given confidence to the assignment of part of the 3 A electron density map to the anticodon region of the molecular structure of yeast tRNAPhe.

Separation of two initiator transfer RNAs from E. coli.

Two classes o f methionine-accepting transfer R N A have been characterised from the cytoplasm o f mammalian cells. As in other eukaryotic systems, one o f these ( tRNA Met) is found to donate methionine into the internal posttions o f protein cimins, whereas the other, the initiator (tRNA~-~et), acts as a specific donor for the N-terminal methionine residue [band in newly synthesized polypeptides [ 1 3 ] . MethionyltRNA~ let in the cytoplasm 0feukaryotes , unlike that orE. col[ and mitochondria, does not need to be formylated before it can donate methionine into the N-ternfinal positions o f proteins. We are attempting to relate the structure o f a eukaryotic initiator tRNA to its role in the cell with ~ e aim o f explaining the differences in its be!tar[our front the bacterial initiator tRNA. At present we are determining the nucleotide sequence of the tRbrA~ et from m o u ~ P3 myeloma ceils. Very recently the nucleotide sequence o f the first eukaryotic initiator tRNA has been determined by Simsek and Ra jBhanda~ [4] . This yeast tl~NAf Met has a feature distinct from aU other tRNAs o f known sequences functioning in protein biosynthesis: the common T G p T p ~ p C p sequence found in loop IV of the cloverleaf structure it replaced in yeast tRNA~ tot by the sequence G p A p U p C p . Now we have also found the latter sequence in a mammalian initiator ti~NA replacing the common sequence confirming the notion that this region o f the molecule is concerned with the special initiation role. 2. Methods and results

Inhibition of magic spot formation by L-,1-tosylamido-2-penylethyl chloromethyl ketone

Recently the sequence of the bacterial initiator tRNA, tRNAfMet , was elucidated [ 11. Durin the course of the sequence work a minor tRNA B & et was detected having an A residue instead of a 7Me residue at position 3 1 numbered from the amino acid acceptor end. The relative proportions of the two tRNAr species were estimated as approximately 75% with a 7MeG and 25% with an A. Evidence was further presented to show that this base change was probably the only difference between the two tRNAs. This communication describes the partial separation of these two tRNAf species and demonstrates that the two species have identical coding properties. tRNA, labelled with s2P was first separated by chromatography on DEAE Sephadex as desc&d elsewhere [2]. Subsequently the purified tRNAf species were subjected to chromatography on benzoylated DEAE cellulose [3]. Fig. 1 shows the result of such an experiment. The first eluted peak (A) containing the tRNAf activity is asymmetrical, suggesting a partial separation of two isoaccepting species, tRNAget and tRNAFt. For this reason tRNApf was isolated from fractions at the indicated positions of the elution profile. After their isolation samples from the two fractions were digested with Tl or pancreatic ribonuclease. The resulting enzymic digests were then fractionated by the usual two-dimensional system [4] using 7% formic acid in the DEAE paper dimension. Fig. 2 shows a comparison of the Tl ribonuclease “fingerprints” of the respective tRNAf fractions. Fig. 3 shows a similar comparison of the pancreatic ribonuclease “fingerprint”. It is evident that tRNAet from fraction 1 contains relatively small amounts of the ‘i’MeGcontaining sequence (about 30%), while having a high proportion of the corresponding A-containing sequence (about 70%). The tRNA,, Met from fraction 2, on the