Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.     

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5′-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3′ side, which contains the Shine–Dalgarno sequence. Here, we show by in vitro gp43–RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine–Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator’s hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5′-AC-3′ in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.     

Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly.

The groE protein, which is involved in the morphogenesis of several bacteriophages, was isolated using a hybrid bacteriophage λ strain which overproduces it. The protein was characterized using biophysical methods, electron microscopy and digital image processing. We postulate that the gp groE complex contains 14 subunits in a cylindrical aggregate with 7-fold rotational symmetry. Possible mechanisms are discussed for the action of this complex in phage morphogenesis.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T = 9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.