Feldmann, H.

Gruhl, H.

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Volume 57, number 2 FEBS LETTERS September 1975 
THE PRIMARY STRUCTURE OF A NON-INITIATING METHIONINE 
SPECIFIC tRNA FROM BREWER’S YEAST 
H. GRUHL and H. FELDMANN 
Institut fiir Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitiit Miinchen, 
8 Miinchen 2, GoethestraJe 33, West Germany 
Received 30 June 1975 
1. Introduction 
Methionine specific tRNAs, because of a number 
of unique features due to their role in protein biosyn- 
thesis, are a most interesting family of isoacceptors. 
In any organism so far investigated, at least two species 
of tRNAMet are present, the structures of which have 
been adapted for their participation in peptide chain 
initiation (tI2NAy) and peptide chain elongation 
(tRNAMet); for a recent review see [l] . 
Themprimary structures of a variety of methionine 
specific tRNAs (tRNAp [2] and tRNAp [3] from 
E. cd, tRNAref from baker’s yeast [4], tRNA?et 
[5,6] and one tRNAp [7] from a mammalian source) 
have been elucidated. The information obtained has 
been discussed with regard to structure-function rela- 
tionship, e.g. [5,6] and evolutionary aspects e.g. [6]. 
We reported earlier [8] , that brewer’s yeast contains 
three major species of tRNAMet, one of which 
(tRNApet = tRNApef) has been shown to function . 
as an initiator tRNA [8], while the two others 
(tRNApet and tRNAyet) are involved in peptiae 
chain elongation. In contrast, baker’s yeast [9,10] 
and a haploid yeast strain [8] have only one non- 
initiator tRNA, which seems to correspond to 
tRNApt (brewer’s yeast). 
We want to report here on the primary structure 
of tRNApet (brewer’s yeast) which can be considered 
to be a ‘missing link’ for structural comparison within 
the tRNAMet species from E. coli, yeast, and mama- 
lian sources. Some interesting features were found in 
tRNApef (brewer’s yeast) that will be briefly discussed 
with respect to the following topics: (i) sequence homo- 
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logies in tRNAsMet , (ii) synthetase recognition, (iii) 
conservation of structural features in tRNAsMet . 
2. Experimental 
S. cerevisiae, strain C 836, was cultured in a mini- 
mal medium and labeled with [32P] phosphate as 
described in [ 1 l] . One procedure employed in purifi- 
cation of tRNApet was essentially similar to that used 
in the isolation of non-labeled tRNAp* from brewer’s 
yeast [8] . A second, simplified procedure to isolate 
labeled tRNApet was worked out concomitantly 
with the structural work reported here and will be 
detailed in a subsequent publication. In general, the 
methods for determining the nucleotide sequence were 
the standard procedures as described in [ 121 and [ 131. 
Oligonucleotides from partial digestions with RNase 
T1 were resolved by homochromatography [ 131 and 
by two-dimensional gel electrophoresis [ 141. 
3. Results and discussion 
3.1. Primary structure of tRNApt 
The primary structure of tRNApet (brewer’s yeast), 
drawn as a clover-leaf, is shown in fig.l(A). The nucleo- 
tide sequence was derived by some analyses on non- 
labeled tRNA, and mainly be the 32P-fingerprinting 
method [ 121. Complete overlaps in the nucleotide 
sequence could be deduced from the oligonucleotides 
of digestions with RNase Tl and RNase A, respective- 
ly, and from 18 products (out of 29 analyzed) of 
145 
Volume 57. number 2 FEBS LETTERS September 1975 
partial digestions with RNase T1. The details of 
sequencing will be fully documented in a further 
publication. 
tRNApet (brewer’s yeast) is 76 nucleotides in 
length, possessing 13 modified nucleosides (4 $, 2 D, 
T, m’A, m2G, m:G, m7G, m5C, and t6A). In the 
sequence analyses most of these modifications were 
found to be complete except the following: D in 
position 47 was only present in 75%, the rest was 
found to be U; the bases in miGp$p (positions 26 
and 27) were found to be modified to only 50%, the 
rest was GpUp. 
Fig.]. Comparison of the cloverleaf structures of methionine 
specific tRNAs. (A) Primary structure of tRNAFet (brewer’s 
yeast). Stems and loops are numbered according to the no- 
menclature used in [ 15 I. Boxes: homologies with tRNAfMet 
(yeast) (cf. also text). (B) Homologies of tRNAyet (brewer’s 
yeast) with the non-initiator methionine specific tRNAs from 
mammalian sources [ 7,171 (large capitals), and E. Coli 131 
(dotted boxes). Small capitals refer to nucleosides present in 
the E. coli and the brewer’s yeast tRNA in these positions; 
C* can be Cm in tRNAyet 171, A* is m’A in tRNAyet 
(brewer’s yeast) and tRNAyet 171. G* is Cm and U* is 
s4 U in tRNAze’ (E. co/i) [ 3 ] CC) Iiomologies in all meth- 
ionine specific tRNAs sequenced. Asterisks identify nucleo- 
sides which are differently modified. Constant nucleotides 
in all tRNAs are put in circles. The dotted lines connect 
co-ordinate bases [ 2 11. 
3.2. Comparison of tRNAMet sequences from yeast 
The sequence of tRNAklet (brewer’s yeast) is largely 
different from that of the initiating tRNAMet (brewer’s 
yeast), which was found to have the same nucleotide 
sequence [16] as that from baker’s yeast [4] (fig.l(A)). 
Sequence homologies in tRNApt and tRNApet are 
present in only 42 positions, these including a number 
of nucleotides in the D-loop (I) and in the D-stem (b), 
the sequence -m7G-D-mSC- in the extra loop (III), 
the last four nucleotides at the acceptor end, and the 
positions of m2G and m;G. Many of these similarities, 
however, reside in positions in which certain nucleo- 
146 
Volume 57, number 2 FEBS LETTERS September 1975 
sides were found to be constant in all tRNAs. Also, 
tRNApet and tRNA let (E. coli) were found to have 
no greater similarities with each other in sequence 
than with other tRNA sequences from the same or- 
ganism [3]. The most obvious dissimilarities between 
tRNAyet and tRNA yet (in brackets) are: the D-loop 
(I) contains 8 (7) nucleotides, there is no unusual 
dinucleotide (G*pA*p) [4] in stem (e), loop (IV) 
has the ‘normal’ construction Tp$pCpGp (ApUpCpGp), 
there are two pseudouridine nucleotides in opposite 
positions at the base of stem (c). 
3.3. Comparison of sequences of non-initiator tRNAMet 
species from different organisms 
Fig.l(B) shows the sequence homologies between 
tRNApet (brewer’s yeast), the tRNAEet species of ,5’. 
coli [3] and tRNAEf species from two mammalian 
sources [7,17]. The latter two tRNAs seem to have 
identical sequences [7 ] like the initiator tRNAs of 
mammalian origin [6]. It is obvious that the sequence 
variations between E. coli and yeast tRNAstet are 
greater than between yeast and mammalian tRNAsp. 
In the first case, the following similarities are found: 
the end of the acceptor stem (a), the complete D-stem 
(b), some of the positions within the D-loop (I) (al- 
though tRNAtet (E. coli) possesses 9 nucleotides in 
this loop instead of 8 in eukaryotic tRNAszet), the 
anticodon loop (II), and most of the T$C-loop (IV). 
The eukaryotic tRNAsm Met display great similarities: 
the anticodon arms and the extra loops (III) are iden- 
tical, only one position (20) is differing in the D-loop 
(I) and one (59) in the T$C-loop (IV). Altogether 5 
base pairs are different in the acceptor stem (a), in 
the D-stem (b), and in the T$C-stem (e). A unique 
feature is the occurrence of two pseudouridine 
nucleotides in opposite positions at the base of 
the anticodon stem (c), which so far were only 
found in the eukaryotic non-initiator tRNAsMet 
]7,171. 
3.4. Recognition sites and evolutionary aspects 
It has been shown that E. coli methionyl tRNA 
synthetase can charge not only the homologous 
tRNAsMet but also the tRNAMef species from yeast 
e.g. [s-Id] and eukaryotic initiator tRNAs [6,7,18, 
191, exceptions being the non-initiating tRNAsMef 
from reticulocytes [ 181, wheat germ [ 191, and from 
rabbit liver, e.g. [ 171 . Extended studies on heterologous 
charging of tRNAsMet with methionine specific syn- 
thetases from eukaryotic sources have not been re- 
ported. 
However, from a comparison of the primary struc- 
tures of all tRNAsMet so far known (fig.lC) it becomes 
apparent, that these do not exhibit common features 
except the following: (i) the constant nucleotides 
found in similar positions in all tRNAs, (ii) nearly 
the whole anticodon loop, (iii) the fourth nucleotide 
from the 3’-end (being Ap), (iv) two base pairs (in- 
cluding the Levitt base pair [20]), and m’G. 
It therefore seems to be justified to conclude that 
in the case of the methionine specific tRNAs from a 
number of different organisms charged with E. coli 
methionyl-tRNA-synthetase no homologous sequences 
(except part of the anticodon loop and the ApCpCpA- 
end) could be involved in recognition. 
It is also obvious from this comparison (fig. 1 C), 
that all tRNAsMet belong to the class I type of &RNAs 
[20] with variations off 1 nucleotide in the D-loop 
(I) and that two out of the three postulated base 
triples [21], namely C13, G22, m’G46 and U8,A14, 
A21 remain constant in this group of tRNAs, while 
the third possible base triple could vary: A9,U12,A23 
(tRNA@’ in E. coli and yeast), G9,G12,C23 or m’G9, 
G12$23 in the initiator tRNAs, and A9,G12$23 (?) 
in tRNAyef ( y I m e oma). Co-ordinate basis of this 
type, however, are not unique to methionine specific 
tRNAs, but are similarly found in many other specific 
tRNAs of the class I type in different organisms [21, 
221. It therefore remains to be seen, whether varia- 
tions of such elements determining the tertiary struc- 
ture of tRNA could contribute to specificity in recog- 
nition processes. 
From the comparison of the tRNAyef and tRNApf 
sequences, (especially those from yeast) it is also im- 
possible to detail which features specify whether a 
certain tRNAMet will function in initiation or elonga- 
tion of protein synthesis. 
It has been suggested [7] that the different sequen- 
ces of loop (I) and loop (IV) as well as those of the 
stems (a), (c) and (e) in the mammalian type tRNAsMet 
may be important in discriminating their functions. 
This observation also holds largely for the tRNAsMef 
from brewer’s yeast (see section 3.2. and fig.lA). In 
this context, it is again to be noted that the anticodon 
arm and loop (IV) seem to have been conserved in 
the eukaryotic non-initiator tRNAs (fig. 1 B), so that 
147 
Volume 51, number 2 FEBS LETTERS September 1975 
these two regions might play an important role in 
specifying the function of these tRNAs in peptide 
chain elongation. 
There is further indication that certain features 
of the tRNA sequences in eukaryotes have been 
stabilized during evolution. This has been emphasized 
especially for the initiator tFCNAs [5,6] . A similar 
situation appears to exist for the non-initiator tRNAsMet 
It would be interesting to include in such compa- 
risons also the primary structures of the iso-accepting 
non-initiator tRNAsMet from eucaryotes [7,8]. How- 
ever, the sequences have not yet been determined, 
and it is also unknown why these multiple tRNA 
species do occur. 
It has not escaped our attention that tRNAyet 
(brewer’s yeast) contains the nucleotides found in 
equivalent positions in those tRNAs that are sub- 
strates for homologous or heterologous charging 
with phenylalanine by phenylalanyl tRNA synthe- 
tase from yeast [23] . 
Acknowledgements 
We would like to thank Professor Zachau for his 
interest and discussions. We are grateful to Miss 
B. Beckmann for expert technical assistance. The 
‘Deutsche Forschungsgemeinschaft’ has supported 
this work. 
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148 


The primary structure of a non-initiating methionine specific tRNA from brewer's yeast 

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The primary structure of a non-initiating methionine specific tRNA from brewer's yeast

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