Skip to main content Accessibility help
×
Home
Hostname: page-component-564cf476b6-jjt9s Total loading time: 0.205 Render date: 2021-06-22T21:37:16.366Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "metricsAbstractViews": false, "figures": true, "newCiteModal": false, "newCitedByModal": true, "newEcommerce": true }
RNA RNA

Article contents

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

Published online by Cambridge University Press:  01 November 1998

C. STEFAN VÖRTLER
Affiliation:
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany
OLGA FEDOROVA
Affiliation:
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany Present Address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
TINA PERSSON
Affiliation:
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany
URSULA KUTZKE
Affiliation:
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany
FRITZ ECKSTEIN
Affiliation:
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany
Get access

Abstract

2′-Deoxynucleoside 5′-α-thiotriphosphates have been incorporated randomly, replacing any of the four nucleotides separately and at a low level in Escherichia coli tRNAAsp transcripts. After some tRNAs were charged with the cognate aminoacyl-tRNA synthetase and biotinylated, charged and uncharged tRNAs were separated by binding to Streptavidin. A comparison of the iodine cleavage pattern of charged and uncharged tRNAs indicated positions of 2′-deoxy-phosphorothioate interference with charging. To separate the 2′-deoxy from the phosphorothioate effect, the same sequence of reactions was performed with the corresponding NTPαS. Several positions were identified with a 2′-deoxy or a phosphorothioate effect. tRNAs with single deoxy substitutions at the identified positions were prepared by enzymatic ligation of chemically synthesized halves. The kinetics of charging these tRNAs were determined. The 2′-deoxy effects identified by the interference assay were confirmed, showing a reduction in charging efficiency of between 2.5–6-fold, except for the terminal A76 with a 25-fold reduction. Inspection of the X-ray structure of the tRNA-synthetase complex showed consistency of most of these findings. Critical 2′-deoxy groups are localized mainly on the proposed contact surface with the synthetase or at the interface of the two tRNA domains. The same overall picture emerged for critical phosphorothioates. With the exception of 2′-deoxy-adenosine-containing tRNAs, multiple 2′-deoxy-substituted tRNAs, prepared by ligation of halves, showed a much larger reduction in charging efficiency than the mono-substituted tRNAs, indicating an additive effect.

Type
Research Article
Information
RNA , Volume 4 , Issue 11 , November 1998 , pp. 1444 - 1454
Copyright
© 1998 RNA Society

Access options

Get access to the full version of this content by using one of the access options below.

Send article to Kindle

To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study
Available formats
×

Send article to Dropbox

To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study
Available formats
×

Send article to Google Drive

To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study
Available formats
×
×

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *