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E. coli tRNA Leucine Identity and Recognition Sets

Citation

Tocchini-Valentini, Giuseppe D. (1997) E. coli tRNA Leucine Identity and Recognition Sets. Dissertation (Ph.D.), California Institute of Technology. https://resolver.caltech.edu/CaltechTHESIS:03012017-091934429

Abstract

E. coli contains five different tRNAs which recognize the six leucine codons. These tRNAs are all recognized by the single leucyl-tRNA synthetase (LeuRS). We have used in vitro and in vivo methods to determine the set of identity elements which distinguish the set of leucine tRNAs from all other tRNAs allowing the faithful translation of the leucine codons.

An identity swap experiment has been used to determine which of the nucleotides conserved in all leucine tRNAs are identity elements. In this experiment the identity of an amber suppressor tRNASer was changed completely to leucine. This experiment was effective because the anticodons in tRNASer and tRNALeu are not recognized by their respective synthetases and consequently in both cases tRNAs containing the CUA anticodon required in amber suppressors are fully active.

In its minimal form the Ser-Leu swap required six changes, five of which altered the tertiary structure of the tRNA: the G15-C48 tertiary "Levitt base-pair" in tRNASer was changed to Al5-U48 found in all leucine tRNAs; it was necessary to insert one nucleotide and to delete one nucleotide so as to position the conserved D-loop Gl8, Gl9 nucleotides as they are in all leucine tRNAs; a base was inserted at position 47n between the base-paired extra stem and the T-stem to achieve a configuration found in all leucine tRNAs; in addition it was necessary to change the G73 "discriminator" base in tRNASer to A73, found in all leucine tRNAs. This minimally altered tRNASer inserted exclusively leucine as an amber suppressor and it was an excellent in vitro substrate for LeuRS.

Both tRNASer and tRNALeu are type II tRNAs containing large base-paired extra stem loops. In the case of tRNASer the extra stem loop is a crucial identity element but for tRNALeu earlier in vitro and in vivo experiments had indicated that it is not an identity element. To investigate the role of tRNA tertiary structure in leucine identity we carried out a parallel swap experiment in which the glutamine identity of the amber suppressor tRNASerΔ (in which the type II extra stem loop had been replaced by a consensus type I loop) was converted to leucine. This "type I" swap experiment was also successful both in vivo and in vitro. Interesting differences in the role of conserved leucine base-pairs in the acceptor stems of leucine tRNAs were observed in the two experiments. In the type II swap the conserved acceptor stem bases were not important. In the type I swap their absence had a large effect both in vivo and in vitro. This result indicates that the presence of the extra stem loop in leucine tRNAs has an effect on the tertiary structure of the tRNA. When this structure is altered conserved nucleotides, unimportant in its presence, take on an important role. Possible reasons for this effect are discussed.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Abelson, John N.
Thesis Committee:
  • Unknown, Unknown
Defense Date:19 December 1996
Record Number:CaltechTHESIS:03012017-091934429
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:03012017-091934429
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10082
Collection:CaltechTHESIS
Deposited By: Bianca Rios
Deposited On:01 Mar 2017 17:54
Last Modified:02 Dec 2020 02:35

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