Book/Dissertation / PhD Thesis

FZJ-2017-05677

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Translation Initiation with 70S Ribosomes: A Single Molecule Study

Remes, C. (Corresponding author)FZJ*

2018
Forschungszentrum Jülich GmbH Zentrlabibliothek, Verlag Jülich
ISBN: 978-3-95806-358-7

Please use a persistent id in citations: http://hdl.handle.net/2128/19851  urn:nbn:de:0001-2018120725

Abstract: Until now, three initiation pathways for ribosomal protein synthesis have been described: the 30S-binding mechanism of canonical mRNAs, which assumes that the first step is binding of the small subunit to the start codon of the mRNA with the help of initiation factors, the rare initiation of leaderless mRNAs by full 70S ribosomes, and the 70S-scanning mode through which the second cistron of apolycistronic mRNA is translated. According to the standard model from classical textbook, translation of canonical mRNAs occurs exclusively via the 30S-binding mechanism, which requires dissociation of subunits. In contrast to this, we demonstrated here using an $\textit{in vitro}$ transcription-translation system that canonical mRNAs can be also initiated by full 70S ribosomes, without dissociation. The standard and non-standard initiation events were quantified with single molecule experiments, since only by tracking one particle at a time one can identify and count parallel mechanistic initiation pathways. To perform single molecule experiments, the sample preparation had first to be optimized by establishing the protocols for reassociation of ribosomes, biotinylation and labeling. The reassociation procedure involves purification of tightly-coupled 70S from $\textit{E. coli}$, dissociation into subunits and reassociation to form 70S monosomes, and it leads to a highly pure sample, in which the endogenous bacterial mRNAs and factors are discarded. Concerning the biotinylation, two approaches are presented: onthe one hand, $\textit{in vivo}$ biotinylation of the uL4 ribosomal protein, and on the other hand, binding of a biotinylated oligonucleotide to an RNA loop engineered on the small subunit. The first approach lead to a 1/4 decrease in activity of the modified ribosomes, in contrast to the second method, which did not affect the activity of the ribosomes. In addition to this, ribosomes were fluorescently labeled for single molecule experiments, using two strategies: unspecific labeling of accessible lysines with NHSdyes and labeling via oligo nucleotide binding on engineered loops of rRNA. In both cases, the samples were characterized in regard to the labeling ratio, presence of aggregates and free dye, and activity. The first method allowed multilabeling of ribosomes, but a 1/3 decrease in activity was observed, while the second approach yielded site-specifically single labeled ribosomes with an activity comparable to the wild type. The second step was improving the stalling properties of the DNA used for GFPem synthesis. A poor stalling efficiency would release the GFPem from the ribosomal complexes, so it would not be detected on a wide field. Moreover, the ribosomal subunits would dissociate, meaning that the 70S which initiated translation would not be identified. Previous studies used the secretion monitor (SecM) arrest sequence to stall ribosome nascent chains. However, the SecM showed unsatisfactory stalling characteristics in our experiments, considering that most of the translated protein was released from the ribosome. Therefore, a new SecM arrest sequence was designed (SecMstr), that was proven to have enhanced stalling properties, achieving an almost absolute stalling of the ribosomes (>95%). The good stalling properties allowed also the quantification of the active fraction of ribosomes in an $\textit{in vitro}$ transcription-translation kit and the average number of productive cycles the ribosomes can perform inthese conditions. [...]

Note: RWTH Aachen, Diss., 2017

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Contributing Institute(s):

1. Molekulare Biophysik (ICS-5)

Research Program(s):

1. 551 - Functional Macromolecules and Complexes (POF3-551) (POF3-551)

Appears in the scientific report 2017

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Database coverage:
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