Studies of the complex of ribosomal protein L1 with its binding site in 23S rRNA: Modification -interference, mutagenesis and crosslinking

2002 2002

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Abstract (summary)

Interaction of ribosomal protein L1 and the 23S rRNA plays an important role in both the structure and biological activity of the Escherichia coli ribosome. We have minimized the binding site for protein L1 on the 23S rRNA to a 32-nucleotide fragment consisting of helix 77, helix 78, and the conserved sequences that interconnect them. Filter-binding, modification-interference and manganese rescue experiments were used do demonstrate (1) that the absence of a 2-OH group at position 2122 can disrupt protein L1-23S rRNA interaction, but only if certain other deoxyribonucleotides are present in the transcript and (2) that the Rp phosphoryl oxygens preceding U2122 and A2176 in the rRNA molecule play a role in Ll-23S rRNA interaction through magnesium ion coordination, possibly by participating in magnesium bridges with the protein. The crystal structure of protein L1 from Thermus thermophilus, which closely resembles Escherichia coli L1, consists of two domains that are divided by a deep cleft. It has been suggested that the binding site for RNA is located within this interdomain cleft. Using site-directed mutagenesis it was possible to identify a cluster of conserved amino acids that are crucial for protein-RNA interactions (F37, D42, T216, G218), as well as several amino acids that help to stabilize the complex (K36, P137, N138, K140, H171, K176, M217). All of the mutagenized amino acids are located on the surface of the interdomain cleft. To orient L1 relative to the RNA, crosslinking studies were performed using photoreactive moieties incorporated into the L1 binding site and protein L1 itself. A 4-thiouridine residue at position 2172 and 2-azidoadenosine ligated to the 3-end of the minimized binding site were shown to form a “zero length” crosslink with protein L1. Whereas the azidophenacyl group attached to position 40 of the protein was demonstrated to map a portion of loop 76/77 on 23S rRNA.

Indexing (details)

Molecular biology;
0307: Molecular biology
0487: Biochemistry
Identifier / keyword
Pure sciences; Biological sciences; Cross-linking; Modification-interference; Mutagenesis; Ribosomal protein L1; Twenty-threeS rRNA
Studies of the complex of ribosomal protein L1 with its binding site in 23S rRNA: Modification -interference, mutagenesis and crosslinking
Drygin, Denis
Number of pages
Publication year
Degree date
School code
DAI-B 63/01, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
9780493525723, 0493525726
Zimmermann, R. A.
University of Massachusetts Amherst
University location
United States -- Massachusetts
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
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