Importance of the leader region of mRNA for translation initiation of ColE2 Rep protein

Translation initiation of mRNA encoding the Rep protein of the ColE2 plasmid required for initiation of plasmid DNA replication is fairly efficient in Escherichia coli cells despite the absence of a canonical Shine-Dalgarno sequence. To define sequences and structural elements responsible for translation efficiency of the Rep mRNA, a series of rep-lacZalpha translational fusions bearing various mutations in the region encoding the leader region of the Rep mRNA was generated and tested for the translation activity by measuring the beta-galactosidase activity. We showed that the region rich in A and U between the stem-loop II structure and GA cluster sequence, formation of the stem-loop II structure, but not its sequence, and the region between the GA cluster sequence and initiation codon are important along with the GA cluster sequence for efficient translation of the Rep protein. The existence of these important regions in the leader region of the Rep mRNA may explain the mechanism of inhibition of the Rep protein translation by an antisense RNA (RNAI), which is complementary to the leader region.     

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.     

Identification of RNA sequences and structures involved in site-specific cleavage of IGF-II mRNAs.

Insulin-like growth factor-II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region (UTR), rendering an unstable 5' cleavage product containing the coding region and a very stable 3' cleavage product of 1.8 kb consisting of the 3'-UTR sequence and the poly(A) tail. Previously, it was established that two widely separated elements in the 3'-UTR (elements I and II), that can form a duplex structure, are necessary and sufficient for cleavage. To further investigate the sequence and secondary structure requirements for cleavage, we have introduced a number of mutations around the cleavage site and assayed their effects on cleavage. Several recognition determinants involved in the endonucleolytic cleavage of IGF-II mRNAs were identified. Mutational analysis around the cleavage site revealed that cleavage is sequence specific and that the cleavage site must be in a single-stranded conformation to allow efficient cleavage. In addition, we have identified an accessory protein that specifically interacts with a stem-loop structure located 133 to 73 nt upstream of the cleavage site.     

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.     

Decreasing the distance between the two conserved sequence elements of histone pre-messenger RNA interferes with 3' processing in vitro.

Histone mRNA 3' end formation requires the presence of two cis-acting conserved sequence elements: a stem-loop structure upstream from the site of cleavage and a purine-rich region downstream from the site of cleavage called the histone downstream element (HDE). Possible interactions between these two elements and their respective binding factors were investigated by a series of deletions (1-7 nt) in the region between the two. The efficiency of processing decreased as the stem-loop and the HDE were moved closer together. In contrast with the documented ability of the U7 snRNP to direct cleavage at a fixed distance from the HDE in insertion mutants (Scharl & Steitz, 1994), all deletion substrates for which processing was observed were cleaved at or 1-nt upstream from the wild-type site. The reason for the inability of the system to cleave closer to the stem-loop remains unclear, but the removal of stem-loop binding protein(s) (SLBP) did not activate upstream cleavage events. Thus, although the processing machinery measures the distance between the cleavage site and the HDE of mammalian histone pre-mRNAs, there is a barrier limiting how far upstream cleavage can occur. These data allow a reevaluation of the sites of 3' end processing in known histone pre-mRNAs.     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.     

Ribokinase from Escherichia coli K12. Nucleotide sequence and overexpression of the rbsK gene and purification of ribokinase

The nucleotide sequence of a 1455-base pair TaqI-HinfI fragment of the rbs operon of Escherichia coli K12 has been determined. It includes the 3' terminus of rbsB (the gene for ribose-binding protein) and the entire rbsK gene, encoding ribokinase. Potential consensus promoter sequences and a stable stem-loop structure are present in the rbsB-rbsK intercistronic region. The regulatory significance of these sequence features is discussed with respect to the rbs operon. rbsK has been cloned downstream from the Serratia marcescens trp promoter on a multicopy plasmid. Cells harboring this plasmid, when grown on minimal ribose plus ampicillin, express ribokinase at the level of 2% of the soluble protein, and induction with indoleacrylic acid raises ribokinase levels another 8-fold. Ribokinase has been purified to homogeneity (216 mumol/min/mg) from a strain harboring this plasmid. Protein sequence analyses of peptides generated by cyanogen bromide cleavage and o-iodosobenzoic acid cleavage confirmed the translation initiation site and the reading frame of the DNA sequence. Amino acid compositions of native ribokinase and the C-terminal dodecapeptide agree with the predicted amino acid compositions, confirming the accuracy of the DNA sequence and the translation termination site.     

Stability of RNA stem-loop structure and distribution of non-random structure in the human immunodeficiency virus (HIV-I).

The stability of potential RNA stem-loop structures in human immunodeficiency virus isolates, HTLV-III and ARV, has been calculated, and the relevance to the local significant secondary structures in the sequence has been tested statistically using a Monte Carlo simulation method. Potentially significant structures exist in the 5'non-coding region, the boundary regions between the protein coding frames, and the 3' non-coding region. The locally optimal secondary structure occurring in the 5' terminal region has been assessed using different overlapping segment sizes and the Monte Carlo method. The results show that the most favorable structure for the 5' mRNA leader sequence of HIV has two stem-loops folded at nucleotides 5-104 in the R region (stem-loop I, 5-54 and stem-loop II, 58-104). A large fluctuation of segment score of the local optimal secondary structure also occurs in the boundary between the exterior glycosylated protein or outer membrane protein and transmembrane protein coding region. This finding is surprising since no RNA signals or RNA processing are expected to occur at this site. In addition, regions of the genome predicted to have significantly more open structure at the RNA level correlate closely with hypervariable sites found in these viral genomes. The possible importance of local secondary structure to the biological function of the human immunodeficiency virus genome is discussed.     

Essential elements in the coding region of mRNA for translation of ColE2 Rep protein

Translation initiation of mRNA encoding the plasmid-specified initiator protein (Rep) required for initiation of the ColE2 plasmid DNA replication is fairly efficient in Escherichia coli despite the absence of a canonical Shine-Dalgarno sequence. Although a GA cluster sequence exists upstream the initiation codon, its activity as the SD sequence has been shown to be very inefficient. Deletion analyses have shown that there are sequences important for the Rep translation in the regions upstream the GA cluster sequence and downstream the initiation codon. To further define regions important for translation of the Rep mRNA, a set of the ColE2 rep genes bearing single-base substitution mutations in the coding region near the initiation codon was generated and their translation activities examined. We showed that translation of the Rep mRNA was reduced by some of these mutations in a region ranging at least 70 nucleotides from the initiation codon in the coding region, indicating the presence of translation enhancer(s) outside the translation initiation region which is covered by the ribosome bound to the initiation codon. Some of them seem to be essential and specific for translation of the ColE2 Rep mRNA due to the absence of a canonical SD sequence.     

Structural requirements for efficient translational frameshifting in the synthesis of the putative viral RNA-dependent RNA polymerase of potato leafroll virus.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).