Mutational analysis of Brh2 reveals requirements for compensating mediator functions

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.     

In vitro selection of halo-thermophilic RNA reveals two families of resistant RNA

The "RNA world" hypothesis proposes that early in the evolution of life, RNA was responsible both for the storage and transfer of genetic information and for the catalysis of biochemical reactions. One of the problems of the hypothesis is that RNA is known to be temperature sensitive. Nevertheless, different types of sequences with a thermostable phenotype may exist. In order to test this possibility, we applied an in vitro evolution method (SELEX) to isolate RNA molecules that are resistant at high temperatures (80 degrees C for 65 h) and high salt concentrations (2 M NaCl). The sequences of the resulting cloned halo-thermophilic RNAs can be grouped in two families (I and II) possessing very different thermal and chemical stabilities and very different secondary structures. The selected RNA molecules illustrate two different possibilities leading to thermal resistance which may be related to primitive conditions. We propose that members of family I constitute a good means of storing sequence information while members of family II are less efficient but replicate faster in early steps of the SELEX. These selected RNA behaviors may be related to primitive conditions and could allow to define limits for survival, and demonstrate that what is at stake for RNA molecules, as for living organisms, is survival and reproduction.     

Stepping transfer messenger RNA through the ribosome

tmRNA (transfer messenger RNA) is a unique molecule used by all bacteria to rescue stalled ribosomes and to mark unfinished peptides with a specific degradation signal. tmRNA is recruited by arrested ribosomes in which it facilitates the translational switch from cellular mRNA to the mRNA part of tmRNA. Small protein B (SmpB) is a key partner for the trans-translation activity of tmRNA both in vivo and in vitro. It was shown that SmpB acts at the initiation step of the trans-translation process by facilitating tmRNA aminoacylation and binding to the ribosome. Little is known about the subsequent steps of trans-translation. Here we demonstrated the first example of an investigation of tmRNA.ribosome complexes at different stages of trans-translation. Our results show that the structural element at the position of tmRNA pseudoknot 3 remains intact during the translation of the mRNA module of tmRNA and that it is localized on the surface of the ribosome. At least one SmpB molecule remains bound to a ribosome.tmRNA complex isolated from the cell when translation is blocked at different positions within the mRNA part of tmRNA.     

Mutational analysis of 3' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In all of the stable lines, the point mutations affected splicing efficiency in this region.     

Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.     

Structural dynamics of ribosomal RNA during decoding on the ribosome

Decoding is a multistep process by which the ribosome accurately selects aminoacyl-tRNA (aa-tRNA) that matches the mRNA codon in the A site. The correct geometry of the codon-anticodon complex is monitored by the ribosome, resulting in conformational changes in the decoding center of the small (30S) ribosomal subunit by an induced-fit mechanism. The recognition of aa-tRNA is modulated by changes of the ribosome conformation in regions other than the decoding center that may either affect the architecture of the latter or alter the communication of the 30S subunit with the large (50S) subunit where the GTPase and peptidyl transferase centers are located. Correct codon-anticodon complex formation greatly accelerates the rates of GTP hydrolysis and peptide bond formation, indicating the importance of crosstalk between the subunits and the role of the 50S subunit in aa-tRNA selection. In the present review, recent results of the ribosome crystallography, cryoelectron microscopy (cryo-EM), genetics, rapid kinetics and biochemical approaches are reviewed which show that the dynamics of the structure of ribosomal RNA (rRNA) play a crucial role in decoding.     

Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection and DNA transfer

The F-pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F-pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F-pilus tip, and the RNA phages, R17 and Qbeta, which attach to different sites exposed on the sides of the pilus. R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells. New and existing mutants of traA combined with natural variants of F-pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F-pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45-60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.     

Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity

An RNA-dependent RNA polymerase (replicase) activity that specifically copies brome mosaic virus (BMV) RNAs in vitro can be prepared from BMV-infected barley leaves. The signals directing complementary (minus) strand synthesis reside within the 3' 134-nucleotide-long tRNA-like structure that is common to each of the virion RNAs. By studying the influence of minus strand synthesis of numerous mutations introduced throughout this region of the RNA, we have mapped in detail the sequence and structural elements necessary for minus strand promoter activity. Sequence alterations (either substitutions or small, structurally discrete deletions) in most parts of the tRNA-like structure resulted in decreased minus strand synthesis. This suggests that BMV replicase is a large enzyme, possibly composed of several subunits. The lowest activities, 5 to 8% of wild type, were observed for mutants with substitutions at three separate loci, identifying one structural and two sequence-specific elements essential for optimal promoter activity. (1) Destabilization of the pseudoknot structure in the aminoacyl acceptor stem resulted in low promoter activity, demonstrating the importance of a tRNA-like conformation. (2) Substitution of the C residue adjacent to the 3' terminus resulted in low promoter activity, probably by interfering with strand initiation. (3) The low activities resulting from substitutions and a small deletion in arm C suggest this region of the RNA to be a major feature involved in replicase binding. In particular, nucleotides within the loop of arm C appear to be involved in a sequence-specific interaction with the replicase.     

Messenger RNA movement on the ribosome

Recent X-ray and cryo-EM studies of 70S ribosome complexes containing different types of messenger RNAs (mRNA) and transfer RNA (tRNA) have been reviewed. Changes of the mRNA path on the ribosome at initiation and elongation states have been described. Authors suggested, that the specific region of ribosomal 30S subunit ("platform") is a ribosome binding site of regulatory domains of mRNA which locates on the non-translated 5'-end of the mRNA.     

Messenger RNA movement on the ribosome

The review summarizes the recent structural data obtained for 70S ribosome complexes with various mRNAs and tRNAs by X-ray analysis and cryoelectron microscopy. The mRNA region interacting with the ribosome at translation initiation and elongation is described. A specific part (platform) of the 30S ribosome subunit was assumed to bind the regulatory elements located in the 5′-untranslated region of mRNA.     

Helix 69 is key for uniformity during substrate selection on the ribosome

Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.     

Mutational analysis of the oxidoreductase ERp57 reveals the importance of the two central residues in the redox motif

The oxidoreductase ERp57 is involved in the formation and breaking of disulfide bonds in assembling proteins within the environment of the endoplasmic reticulum. Site-directed mutants of the redox-active Cys-Gly-His-Cys motif within an isolated ERp57 sub-domain have been studied. Whereas mutation of either cysteine residue abolished reductase activity, substitution of the central residues resulted in retention of partial activity. Alkylation studies indicated that the central residue mutants retained the normal disulfide bond in the motif, whereas this disulfide bond became more resistant to reduction following addition of a third residue into the redox motif, demonstrating an optimum spacing within the redox-active motif of ERp57.     

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.     

Mutational analysis reveals Escherichia coli oriC interacts with both DnaA-ATP and DnaA-ADP during pre-RC assembly

Prior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three- to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites. Increasing the number of discriminatory sites by replacing R5M with I2 inactivated extrachromosomal oriC function. Mutants with no discriminatory sites perturbed host growth and rapidly replaced wild-type chromosomal oriC, but normal function returned if one I-site was restored at either the I2, I3 or R5M position. These observations are consistent with assembly of E. coli pre-RC in vivo from mixtures of DnaA-ATP and DnaA-ADP, with I-site interactions coupling pre-RC assembly to DnaA-ATP levels.     

Mutational analysis of the hepatitis C virus RNA helicase.

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.     

EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome.

Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion. The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation. In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation. We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm.