Asymmetric maturation of a dimeric transfer RNA precursor

Six of the eight transfer RNAs coded by bacteriophage T4 are synthesized via three dimeric precursor molecules. The sequences of two of these have been determined. Both of these precursors give rise to equimolar amounts of the cognate tRNA molecules in vivo. In contrast, even in wild-type infections, tRNA^Ile is present in ≤ 30% the amount of tRNA^Thr, with which it is processed from a common dimeric precursor.We have now determined the sequence of this dimer. In addition to the nucleotides present in tRNA^Thr and tRNA^Ile, it contains nine precursor-specific residues, located at the 5′ and 3′ termini and at the interstitial junction of the two tRNA sequences. While the three dimers share the majority of structural features in common, pre-tRNA^{Thr + Ile} is the only case in which an encoded tRNA 3′ -C-C-A terminus is present in the interstitial region.The processing of this dimer in various biosynthetic mutants has been analyzed in vivo and in vitro and shown to be anomalous in several respects. These results suggest that the apparent underproduction of tRNA^Ile can be explained by a novel processing pathway that generates a metabolically unstable tRNA^Ile product. Data from DNA sequence analysis of the T4 tRNA gene cluster (Fukada & Abelson, 1980) support the conclusion that the asymmetric maturation of this precursor is a consequence of the unique disposition of the -C-C-A sequence. These results argue that gene expression can be modulated at the level of RNA processing. The biological significance of this phenomenon is discussed in relation to evidence that tRNA^Ile has a unique physiological role.     

Self cleavage of a precursor RNA from bacteriophage T4

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.     

Three suppressor forms of bacteriophage T4 leucine transfer RNA

Three suppressor forms of bacteriophage T4 leucine transfer RNA were isolated and characterized. One suppresses U-A-G mutations, another suppresses U-A-G and U-A-A mutations, while the third suppresses U-G-A mutations. Each suppressor specifies a new anticodon sequence in leucine transfer RNA. Whereas wild-type leucine transfer RNA has the anticodon sequence N-A-A (N is a modified U), the suppressor forms have C-U-A, N-U-A or N-C-A, respectively.     

Assembly-dependent conformational changes in a viral capsid protein. Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.     

Activation of a silent gene is accompanied by its demethylation

The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation.     

The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation

A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

Location and orientation of the chromophore in bacteriorhodopsin: Analysis by fluorescence energy transfer

The spatial location and orientation of the retinal chromophore in bacteriorhodopsin were estimated from a fluorescence energy transfer study. The energy donor used in this study was a fluorescent retinal derivative, which was obtained by partial reduction of the purple membrane with sodium borohydride, and the energy acceptor was the native chromophore remaining in the same membrane. Since bacteriorhodopsin forms a two-dimensional crystal with P₃ symmetry in the purple membrane, and the membrane structure is maintained after the reduction, the rate of energy transfer from a donor to any acceptor existing in the same membrane can be calculated as a function of the location and orientation of the chromophores in the unit cell. Quantitative analyses of the fluorescence decay curve and the quantum yield, with various extents of reduction, enabled us to determine the most probable location and orientation. The result suggested that the chromophore was situated near the centre of the protein in such an orientation that the dipole-dipole interaction with neighbouring chromophores was close to minimum.     

Genes and pseudogenes for human U2 RNA: Implications for the mechanism of pseudogene formation

Three loci, designated U2/4, U2/6 and U2/7, which contain sequences related to human U2 RNA, have been studied. The U2/6 locus contains a tandem array of bona fide U2 genes. U2/4 and U2/7, in contrast, contain pseudogenes whose sequences deviate significantly from that of mammalian U2 RNA. The two pseudogenes appear to have been created by different mechanisms. The sequences that flank the pseudogene in the U2/4 locus lack homology to the corresponding sequences in functional human U2 genes, except for 10 base-pairs immediately following the 3′ end. The conserved 3′-flanking segment is homologous to those nucleotides that are present in a U2 RNA precursor. No direct repeats flank the pseudogene in the U2/4 locus. The observations thus suggest that a complementary DNA copy of the U2 RNA precursor was inserted into a blunt-ended chromosomal break to generate the U2/4 locus.The U2/7 locus, in contrast, revealed flanking sequence homology when compared to functional U2 genes, both on the 5′ and 3′ sides of the pseudogene. The homology was interrupted on both sides by repetitive sequences belonging to the Alu family. On the 5′ side the homology continues beyond the Alu repeats whereas on the 3′ side it ends precisely at the Alu repeat. This Alu repeat is inserted in a region where a homocopolymeric region of alternating C and T residues is located in functional U2 loci. The observed organization of the U2/7 locus suggests that a previously functional U2 locus was invaded by Alu repeats and subsequently accumulated base substitutions to become a pseudogene.     

The effect of dietary fat on the activity,content, rates of synthesis,and degradation and translation of messenger RNA coding for malic enzyme in mouse liver

The specific activity (units activity/mg cytosolic protein) of malic enzyme was found to be three-fold higher in the livers of mice fed a semipurified diet containing 50% ($\text{w}\text{w}$) glucose and 15% ($\text{w}\text{w}$) saturated and monounsaturated but no polyunsaturated fat (hydrogenated cottonseed oil) over an 11-day period than in the livers of mice fed a standard laboratory mouse chow (Purina) diet. In contrast, when other lab chow-fed mice were fed an isocaloric diet containing 15% ($\text{w}\text{w}$) polyunsaturated fat (corn oil), no change in the specific activity of malic enzyme occurred over a similar period of time. Rocket immunoelectrophoresis performed on cytosols from both dietary groups demonstrated that the livers of mice consuming the hydrogenated cottonseed oil diet contained approximately three times more malic enzyme protein than did the livers from the corn oil-fed animals. In mice pulse-labeled with l-[4,5-³H]leucine, the rate of hepatic malic enzyme synthesis (relative to that for total protein) was approximately twofold greater in the hydrogenated cottonseed oil-fed mice than in their corn oil-fed counterparts whereas the rate of hepatic malic enzyme degradation was similar for both groups. Immunotitration of liver malic enzyme from hydrogenated cottonseed oil-fed and corn oil-fed mice revealed identical equivalence points, demonstrating that the catalytic efficiency of mouse liver malic enzyme had not been affected by the type of dietary fat administered. When total liver RNA, isolated from the hydrogenated cottonseed oil- and the corn oil-fed animals, was translated in cell-free translation systems (wheat germ extract and reticulocyte lysate) we found that both dietary treatments had resulted in an increase in the activity of malic enzyme messenger RNA. Furthermore, there were no significant differences between the two dietary groups in this regard. These results suggest that hepatic malic enzyme specific activity in high-carbohydrate polyunsaturated fat-fed mice is regulated principally by dietary-induced changes in the rate of enzyme synthesis and not by the activity of messenger RNA coding for the enzyme.