Useful 'junk': Alu RNAs in the human transcriptome.

Alu elements are the most abundant repetitive elements in the human genome; they have amplified by retrotransposition to reach the present number of more than one million copies. Alu elements can be transcribed in two different ways, by two independent polymerases. 'Free Alu RNAs' are transcribed by Pol III from their own promoter, while 'embedded Alu RNAs' are transcribed by Pol II as part of protein- and non-protein-coding RNAs. Recent studies have demonstrated that both free and embedded Alu RNAs play a major role in post transcriptional regulation of gene expression, for example by affecting protein translation, alternative splicing and mRNA stability. These discoveries illustrate how a part of the 'junk DNA' content of the human genome has been recruited to important functions in regulation of gene expression.

Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis.

The kidney glomerulus plays a crucial role in blood filtration but the molecular composition and physiology of the glomerulus is not well understood. We previously constructed and large-scale sequenced four mouse glomerular expressed sequence tag (EST) libraries from newborn and adult mouse glomeruli. Here, we compared glomerular EST profiles with whole kidney EST profiles, thereby identifying 497 transcripts corresponding to UniGene clusters that were glomerulus-enriched, that is expressed more abundantly in glomeruli than in whole kidney. These include several known protein-coding glomerulus-specific transcripts critical for glomerulus development and function, but also a large number of gene transcripts, which have not previously been shown to be expressed in the glomerulus, or implicated in glomerular functions. We used in situ hybridization to demonstrate glomerulus-specific RNA expression for six novel glomerular genes and the public Human Protein Atlas to verify glomerular protein expression for another two. The higher mRNA abundance for the eight genes in glomeruli compared with whole kidney was also verified by Taqman quantitative polymerase chain reaction. We surmise that the further characterization of these genes and proteins will increase our understanding of glomerular development and physiology.

Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells.

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2(+) and pmc3(+) have been shown to be nonessential genes. The data suggest that S. pombe and S. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10 essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.

Mediator--a universal complex in transcriptional regulation.

The Mediator complex is essential for basal and regulated expression of nearly all RNA polymerase II-dependent genes in the Saccharomyces cerevisiae genome. Mediator acts as a bridge, conveying regulatory information from enhancers and other control elements to the promoter. It is now clear that Mediator-like complexes also exist in higher eukaryotic cells and that they have an important role in metazoan transcriptional regulation. However, the exact mechanism of Mediator-dependent transcriptional regulation remains unclear. We review here some recent advances in our understanding of Mediator structure and function. We also discuss a model to account for the functional and evolutionary relationship between yeast and metazoan Mediators. As an appendix to this review, we have created a database, MEDB, in which we have compiled information about all the S. cerevisiae Mediator subunits and their homologues in other eukaryotic cells (http://bio.lundberg.gu.se/medb/).

YidC/Oxa1p/Alb3: evolutionarily conserved mediators of membrane protein assembly.

This review focuses on a novel, evolutionarily conserved mediator of membrane protein assembly in bacteria, mitochondria and chloroplasts. This factor is designated YidC in Escherichia coli, and is localized in the inner membrane. YidC is homologous to Oxa1p in the mitochondrial inner membrane and Alb3 in the chloroplast thylakoid membrane, but does not seem to have a homologue in the endoplasmic reticulum membrane. It has been suggested that YidC operates both as a separate unit and in connection with the SecYEG-translocon depending on the substrate membrane protein that is integrated into the membrane. Mitochondria do not possess a SecYEG-like complex and Oxa1p is thought to form, or to contribute to the formation of, a novel translocon in the mitochondrial inner membrane. Alb3 in the chloroplast thylakoid membrane is, just like YidC and Oxa1p, involved in membrane protein assembly, but only few details are known.

SRPDB (Signal Recognition Particle Database).

Signal recognition particle (SRP) is a stable cytoplasmic ribonucleoprotein complex that serves to translocate secretory proteins across membranes during translation. The SRP Database (SRPDB) provides compilations of SRP components, ordered alphabetically and phylogenetically. Alignments emphasize phylogenetically-supported base pairs in SRP RNA and conserved residues in the proteins. Data are provided in various formats including a column arrangement for improved access and simplified computational usability. Included are motifs for identification of new sequences, SRP RNA secondary structure diagrams, 3-D models and links to high-resolution structures. This release includes 11 new SRP RNA sequences (total of 129), two protein SRP9 sequences (total of seven), two protein SRP14 sequences (total of 10), two protein SRP19 sequences (total of 16), 10 new SRP54 (ffh) sequences (total of 66), two protein SRP68 sequences (total of seven) and two protein SRP72 sequences (total of nine). Seven sequences of the SRP receptor alpha-subunit and its FtsY homolog (total of 51) are new. Also considered are ss-subunit of SRP receptor, Flhf, Hbsu, CaM kinase II and cpSRP43. Access to SRPDB is at http://psyche.uthct. edu/dbs/SRPDB/SRPDB.html and the European mirror http://www.medkem. gu.se/dbs/SRPDB/SRPDB.html

SRPDB (signal recognition particle database).

The signal recognition particle database (SRPDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and organizes SRP-related information about SRP RNA, SRP proteins and the SRP receptor. SRPDB is accessible on the WWW at the URL http://psyche.uthct.edu/dbs/SRPDB/SRPDB.++ +html. A mirror site of the SRPDB is located in Europe at the University of Göteborg, Sweden (http://www.medkem. gu.se/dbs/SRPDB/SRPDB.html ). This release of SRPDB adds 10 new SRP RNA sequences (a total of 117 SRP RNAs), four protein SRP19 sequences (a total of 15), seven new SRP54 (ffh) sequences (a total of 52), and eight sequences of the SRP receptor alpha subunit (FtsY) (total of 36). Sequences are arranged in alphabetical and phylogenetic order and alignments are provided which highlight base paired and conserved regions. SPRDB also provides motifs to find new sequences, a brief introduction to SRP function in protein secretion, numerous SRP RNA secondary structure diagrams, 3-D SRP RNA models, and recently obtained crystal structure PDB coordinates of the human SRP54m domain.

The Signal Recognition Particle Database (SRPDB).

The signal recognition particle database (SRPDB) is located at the University of Texas Health Science Center at Tyler and includes tabulations of SRP RNA, SRP protein and SRP receptor sequences. The sequences are annotated with links to the primary databases. They are ordered alphabetically or phylogenetically and are available in aligned form. As of September, 1998, there were 108 SRP RNA sequences, 83 SRP protein sequences and 28 sequences of the SRP receptor alpha subunit and its homologues. In addition, the SRPDB provides search motifs consisting of conserved amino acid and nucleotide residues, and a limited number of SRP RNA secondary structure diagrams and 3-D models. The data are available freely at the URL http://psyche.uthct.edu/dbs/SRPDB/SRPDB.++ +html

The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA.

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.

Binding of GTP and GDP induces a significant conformational change in the GTPase domain of Ffh, a bacterial homologue of the SRP 54 kDa subunit.

The bacterial Ffh protein is homologous to the SRP54 subunit of the signal recognition particle. Ffh plays a key role in the targeting of proteins to the membrane and it is composed of a N-terminal domain (N), a middle GTPase (G) domain and a C-terminal M domain which has binding sites for SRP RNA and signal peptide. The GTP binding and hydrolysis of Ffh is critical to its function. We have used protease digestion to probe the conformation of the Mycoplasma mycoides Ffh N+G domain. In the absence of nucleotide the protein was comparatively sensitive to protease cleavage and we identified sites particularly prone to cleavage in a region near the C-terminus of the GTPase domain. However, in the presence of GTPgammaS or GDP this region is stabilized and the protein adopts a more ordered structure. The pattern of cleavage with GTPgammaS was indistinguishable from that when GDP was bound, indicating that the conformation of the nucleotide-free form is distinct from that when either GTPgammaS or GDP is bound to the protein. The possible functional role of this significant conformational change is discussed.

The Signal Recognition Particle Database (SRPDB).

This release of the SRPDB (signal recognition particle database, http://pegasus.uthct.edu/SRPDB/SRPDB . html ) adds four SRP RNA sequences (a total of 99 SRP RNA sequences), 23 SRP protein sequences (a total of 63 protein sequences from SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 or SRP72), and, for the first time, sequences of the alpha subunit of the eukaryotic SRP receptor and its homologous bacterial proteins (a total of 21 sequences). Sequences are offered phylogenetically ordered, annotated with links to the primary databases, and in aligned form. Also downloadable are sample SRP RNA secondary structure diagrams, three-dimensional models of representative SRP RNAs, and search motifs.

Ffh and FtsY in a Mycoplasma mycoides signal-recognition particle pathway: SRP RNA and M domain of Ffh are not required for stimulation of GTPase activity in vitro.

Mycoplasma mycoides contains a signal-recognition particle (SRP) composed of an RNA molecule and an SRP54 homologue (Ffh). We have now identified a mycoplasma homologue to the alpha subunit of the mammalian SRP receptor and Escherichia coli FtsY. The protein (MmFtsY) was expressed in E. coli and purified to homogeneity. MmFtsY has a weak intrinsic GTPase activity but GTP hydrolysis was markedly stimulated when it was combined with mycoplasma Ffh (MmFfh) and SRP RNA. Also, in the absence of SRP RNA GTPase activity was significantly enhanced. Furthermore, GTP hydrolysis was stimulated when MmFtsY was combined with the N-terminal GTPase domain (N+G) of MmFfh. These findings indicate that basic features of the GTPase activation mechanism are independent of the C-terminal M domain of the MmFfh protein. We propose that the activation is mediated to a large extent by contacts between the GTPase domains of the mycoplasma Ffh and FtsY proteins and that the contribution of the M domain and SRP RNA in the activation mechanism is mainly for modifying the conformation of the MmFfh GTPase domain.

A 13-kDa protein with a helix-turn-helix motif is encoded by bacterial operons related to the SRP pathway.

We have identified a 13 kDa protein (p13) in Mycoplasma mycoides subsp. mycoides that is encoded immediately downstream of a protein homologous to E. coli FtsY, a protein taking part in the bacterial signal recognition particle (SRP) pathway. The same organisation of the p13 and FtsY genes occurs in Mycoplasma pneumoniae. PCR analysis of different mycoplasma strains revealed the same organisation in strains belonging to the Mycoplasma mycoides cluster of the mycoplasma phylogenetic tree. Searches in sequence databases identified homologues to p13 in Bacillus subtilis and Streptococcus mutans. In these bacteria the p13 protein is encoded by the same operon as a protein homologous to the 54 kDa subunit of SRP. These findings suggest that there is a functional relationship between the p13 protein and the SRP pathway. Sequence analysis of the p13 proteins strongly suggest that they have a helix-turn-helix (HTH) motif, indicating that they are gene regulatory proteins.

The GTPase activity of the Escherichia coli Ffh protein is important for normal growth.

The Escherichia coli (E. coli) Ffh protein is homologous to the 54kDa subunit of the eukaryotic signal recognition particle. We have examined an intrinsic GTPase activity of this protein and have created mutations in one sequence motif (GXXXXGK) of the putative GTP binding site. When glycine-112 was changed to valine (Ffh-G112V), Vmax was reduced to only 4% of the wildtype level. On the other hand, when glutamine-109 was altered to glycine (Ffh-Q109G), the major effect was a 50-fold increase in Km. These results show that the residues Q-109 and G-112 are essential for the binding and hydrolysis of GTP and that they are part of a catalytic site structurally related to that of many other GTPase proteins. Expression of the mutant protein Ffh-G112V in E. coli was highly toxic in the presence of the wildtype protein. In contrast, genetic complementation experiments showed that a viable strain could be constructed where the Ffh-Q109G mutant protein replaced wildtype Ffh. However, expression of the mutant protein had a negative effect on growth rate at 30 degrees C and resulted in elongated cells. These results demonstrate that the GTPase activity of the Ffh protein is required for proper function of the protein in vivo.

Undiscriminating codon reading with adenosine in the wobble position.

To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA. The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated. We have compared the ability of the mutant glycine tRNA1(UCC) and glycine tRNA1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons. The results showed that glycine tRNA1(ACC) unlike glycine tRNA1(UCC) did not fully discriminate between the glycine codons. These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons. In the present paper we give a detailed description of this new in vitro system.

GTPase activity of a bacterial SRP-like complex.

We have recently identified a protein (SRPM54) in Mycoplasma mycoides homologous to SRP54, a subunit of the mammalian signal recognition particle (SRP). This protein forms a complex with a mycoplasma RNA related to the RNA component of SRP. We have now demonstrated that the protein has an intrinsic GTPase activity in vitro and kinetic parameters for the enzymatic reaction have been determined. The GTPase activity was not significantly affected by the presence of the mycoplasma SRP RNA. Different regions of the SRPM54 protein were expressed as recombinant proteins in E. coli and were purified to near homogeneity. On the basis of the properties of these SRPM54 fragments two different functional domains of the protein could be distinguished. An N-terminal part was found to contain the GTPase activity and this domain had approximately the same kinetic properties as the full-length protein. Another domain corresponding to a C-terminal fragment contained the RNA binding activity as shown using an assay based on the retention of RNA-protein complexes to nitrocellulose filters.

A Mycoplasma protein homologous to mammalian SRP54 recognizes a highly conserved domain of SRP RNA.

A protein homologous to SRP54, a subunit of the mammalian signal recognition particle (SRP), was identified in Mycoplasma mycoides. The mycoplasma protein was expressed in E.coli and purified to near homogeneity. It was shown to bind specifically in vitro to a small mycoplasma RNA with structural features related to the RNA component of SRP. These findings provide evidence of a ribonucleoprotein complex in mycoplasma reminiscent of SRP. A part of the RNA was protected from ribonuclease digestion in the presence of the SRP54 homologue. The protected region contains structural elements that have been highly conserved in SRP RNAs during evolution.

Interactions of transfer RNA pseudouridine synthases with RNAs substituted with fluorouracil.

We have previously purified and characterized two different S. cerevisiae enzymes that produce pseudouridine specifically in nucleotide positions 13 and 55, respectively, in their tRNA substrates. The interactions of these enzymes with fluorinated tRNAs have now been studied. Such RNAs were produced by in vitro transcription using as templates synthetic genes that encode variants of a yeast glycine tRNA. RNAs substituted with fluorouracil were found to markedly inhibit pseudouridine synthase activity and the inhibitory effect of a tRNA was to a large extent dependent on the presence of fluorouracil in the nucleotide position where normally pseudouridylation occurs. Pseudouridine synthases were shown to form highly stable, non-covalent complexes with fluorinated tRNAs and we demonstrate that this interaction may be used to further characterize and purify these enzymes. The use of 5-fluorouracil as a cancer therapeutic agent is discussed in relation to our results.

Codon reading properties of an unmodified transfer RNA.

We have previously shown that the Mycoplasma mycoides glycine tRNA (anticodon UCC) effectively reads the codons GGU and GGC in violation of the classic codon reading rules. We have attempted to elucidate what structural elements in this tRNA molecule confer this translational property and in the course of this investigation T7 RNA polymerase transcription of the corresponding gene was used to produce a tRNA devoid of modified nucleosides. Using an in vitro translation system the ability of this tRNA to read the 4 glycine codons (GGU, GGC, and GGG) was tested and it was shown to be as efficient as its normal, fully modified counterpart in the reading of all four codons. This result demonstrates that a tRNA devoid of modified nucleosides is able to efficiently sustain protein synthesis in vitro and, furthermore, that the normal modification pattern of the Mycoplasma glycine tRNA is not essential for the ability of this tRNA to read the glycine codons GGU and GGC effectively.