Phylogenetic relationship of organisms obtained by ribosomal protein comparison

Müller, Eva; Wittmann‐Liebold, Brigitte

doi:10.1007/pl00000578

Cited by 31 publications

(20 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ribosomal protein L6 is one of the most highly conserved ribosomal proteins located at the factor binding site of the large subunit (15,22). The SERF technique identified a consensus sequence U2739-C2789 of domain VI of 23S as the binding site of L6 after four rounds of selection (blue in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…L4 is essential for early events of the 50S assembly (20) and seems to be adjacent to the peptidyl-transferase center (21). L6 is one of the most conserved proteins and is present in ribosomes of all organisms (22) at or near the elongation-factor binding site (15). Furthermore, preliminary experiments demonstrated a low unspecific binding of these proteins to an rRNA fragment pool.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Stelzl

Spahn

Nierhaus

2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Two-thirds of the 54 proteins of the Escherichia coli ribosome interact directly with the rRNAs, but the rRNA binding sites of only a very few proteins are known. We present a method (selection of random RNA fragments; SERF) that can identify the minimal binding region for proteins within ribonucleo-protein complexes such as the ribosome. The power of the method is exemplified with the ribosomal proteins L4 and L6. Binding sequences are identified for both proteins and characterized by phosphorothioate footprinting. Surprisingly, the binding region of L4, a 53-nt rRNA fragment of domain I of 23S rRNA, can simultaneously and independently bind L24, one of the two assembly initiator proteins of the large subunit. Identifying the minimal length of RNA sequences that bind specifically to a protein is a challenge for structural research. The classical approach of digesting an RNA-protein complex with RNase usually does not give the minimal binding region, because the protein(s) within the complex might hinder the access of an RNase. Alternatively, the RNase can cut the RNA within the complex into short sequences that lose the binding capacity. Crosslinking approaches and protection experiments with base modifying reagents indicate vicinity but not necessarily the binding sequence. We decided to exploit the enormous power of in vitro selection methods that can identify the best fitting RNA sequences from about 10 15 variants (SELEX, systematic evolution of ligands by exponential enrichment; ref. 1). However, when randomized sequences are used as starting conditions, affinity selection methods usually do not lead to the naturally occurring binding site for a target as such.Short random fragments from genomic DNA were used to select the DNA binding site of transcription factor IIIA in vitro (2), and a similar idea has been put forward for the selection of protein-nucleic acid interactions (3). In vivo small DNA fragments randomly obtained from 14 genes were identified that stimulate expression of the lacZ gene (4). Expression libraries made of short rRNA sequences have been used to create resistance against antibiotics in vivo (5, 6).The basic idea presented here is that the use of a pool of random fragments from large RNAs directly reveals the native binding site when selected for affinity to a certain protein in vitro. The SELEX variant is abbreviated SERF for selection of random RNA fragments. The principle is shown in Fig. 1. A pool of random rDNA fragments is generated by a random cutting of rDNA. The rDNA fragments are transcribed into RNA in a second step. The advantages of the SERF method are: (i) the selection does not work on just any RNA-protein interactions but rather on the native ones; (ii) the size of the selected rRNA can be chosen and a series of overlapping fragments can reveal the minimal binding site; and (iii) the selection should be fast and efficient because the variability of the pool is low compared with the starting pool used in classical affinity selection experiments.More than two-thirds of t...

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Stelzl

Spahn

Nierhaus

2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In general, structural proteins such as tubulin, actin, and ribosomal proteins show high levels of sequence conservation (15,26,38). In fact, functionality within the Cbf5p-NAP57 family is conserved between widely divergent species; e.g., the Drosophila CBF5 homolog cDNA complements the cbf5⌬ null allele when introduced into yeast on a plasmid and expressed under the control of the yeast ADH1 promoter (8).…”

Section: Discussionmentioning

confidence: 99%

The Yeast Nucleolar Protein Cbf5p Is Involved in rRNA Biosynthesis and Interacts Genetically with the RNA Polymerase I Transcription Factor RRN3

Cadwell

Yoon

Zebarjadian

et al. 1997

Molecular and Cellular Biology

View full text Add to dashboard Cite

Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolarcytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperaturesensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5⌬ null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.Cbf5p of the yeast Saccharomyces cerevisiae was originally isolated as one of the major low-affinity centromeric DNA (CEN) binding proteins (23). CBF5 is an essential gene encoding a highly charged protein with a domain containing ten tandem KKE/D repeats. These repeats are homologous to portions of microtubule-associated proteins 1A and 1B in the domain responsible for microtubule binding (40), and Cbf5p has been shown to bind microtubules in vitro (23). Yeast cells containing C-terminal truncated CBF5 genes delay, with replicated genomes, at the G 2 /M phase of the cell cycle, with the replicated DNA being located at the bud junction (23). Overexpression of Cbf5p suppresses the ndc10-1 temperature-sensitive (ts) mutation in the gene specifying the 110-kDa subunit (Cbf2p/Ndc10p) of the multisubunit yeast centromere DNA binding complex, CBF3, and overexpression of meiosis and centromere regulatory kinase Mck1p suppresses a ts mutation in the gene for either Cbf2p or Cbf5p (22). These genetic interactions support a direct or indirect link between Cbf5p and centromeres. However, both Cbf5p and a homologous protein (NAP57) from rats were found to be nucleolar proteins (21,33). While a functional relationship between a nucleolar protein and centromeres is not obvious, some proteins are associated with both the nucleolus and the centromere in higher eukaryotes. It has been known for some time that centromere autoantigens associate with the nucleolus (43), and experiments with autoimmune sera have also shown that a set of nucleolar proteins and ribonuclear proteins relocate around chromosomes during mitosis (16,17).The nucleolar location of Cbf5p may be indicative of a function unrelated to centromeres and chromos...

show abstract

“…Although protein L2 is among the most conserved ribosomal proteins throughout evolution [9,27], the proteins used in our experiments are rather divergent when compared with their E. coli counterpart. Protein L2 from the halophilic archaebacterium H. marismortui (HmaL2) shows 36 % identical amino acids in comparison with the eubacterial L2 protein, and the human equivalent (HumanL8) only 30 % identity with its E. coli homologue.…”

Section: Discussionmentioning

confidence: 99%

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Ühlein

Weglöhner

Urlaub

et al. 1998

Biochemical Journal

View full text Add to dashboard Cite

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In i o replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and

show abstract

Phylogenetic relationship of organisms obtained by ribosomal protein comparison

Cited by 31 publications

References 50 publications

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

The Yeast Nucleolar Protein Cbf5p Is Involved in rRNA Biosynthesis and Interacts Genetically with the RNA Polymerase I Transcription Factor RRN3

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Contact Info

Product

Resources

About