Late Messenger RNA Research Articles

Regulation of anti-late RNA synthesis in bacteriophage T4: A delayed early control

Bacteriophage T4 make mutually complementary RNA transcripts. The antisense RNA is made in the late region but at early times after infection. It is complementary in base sequence to late messenger RNA and, hence is called anti-late RNA. We have studied some of the physical characteristics and possible regulatory mechanisms involved in the synthesis of these unique early RNA species. Anti-late RNA sediments on 5% to 20% sucrose gradients with an average sedimentation coefficient of 20 to 22 S, comparable to that of late mRNA. The fact that anti-late synthesis becomes refractory to rifampicin before one minute after infection, suggests that it occurs from a class of immediate early promoters in the late regions. Anti-late RNA production is examined under several different conditions of altered early gene expression. In all circumstances where the delayed early gene transcription is altered, anti-late synthesis is also altered. From the data presented here, we postulate that anti-late RNA synthesis is controlled by the same mechanism as that regulating delayed early genes.

Mapping of inverted repeated DNA sequences within the genome of simian virus 40.

Single-stranded, linear DNA of simian virus 40 (SV40) created by denaturing the endonuclease EcoRI- or Hpa II-generated, linear, double-stranded products from form I DNA of SV40 was analyzed for regions of inverted repeated sequences by visualization with the electron microscope. Six hairpin loops were found at positions 0.11-0.30 (two loops forming a "rabbit ears" structure), 0.47-0.52, 0.63-0.68, 0.70-0.76, and 0.90-0.96. The nucleotide sequences within all of these inverted repeats may be related since the looped regions can crosshybridize with one another and, thus, the SV40 genome may contain regions of interspersed repeated and unique sequences. The map positions of the 3' and 5' ends of the early and late messenger RNAs, as determined by others, lie within regions of inverted repeated sequences. Previously recorded recombination events that occurred either within the SV40 genome or between SV40 DNA and other genomes have apparently occurred frequently at positions of inverted repeated sequences within the SV40 DNA.

Mapping of adenovirus messenger RNA by electron microscopy.

Late adenovirus messenger RNA was annealed to complementary regions of partially melted viral double-stranded DNA. The RNA. DNA hybrid regions within the DNA molecules were visualized as loops in the electron microscope. Loops occurred at several regions of the DNA, most frequently, however, at a location near the center of the molecule. This hybridization technique appears well suited for an accurate mapping of messenger RNA, as well as for studies of RNA processing.

Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome

Specific fragments of adenovirus type 2 DNA, generated by cleavage with restriction endonucleases endoR. EcoRI, endoR. HpaI and endoR. HindIII were used in hybridization-mapping experiments. The complementary strands of individual cleavage fragments were separated by the method of Tibbetts & Pettersson (1974). Liquid hybridizations were performed with 32P-labeled separated strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “late” messenger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the 1- and two on the h-strand (h- and l- refer to the strand with heavy and light buoyant density in CsCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locations, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adenovirus type 2 genome.

Early to late switch in bacteriophage T7 development: No translational discrimination between T7 early messenger RNA and late messenger RNA

Cell-free protein-synthesizing systems from uninfected and bacteriophage T7 infected Escherichia coli F− cells synthesized T7 early proteins, T7-specific RNA polymerase and T7 ligase, as well as one of the late proteins, T7 lysozyme, when directed by T7 early mRNA and late mRNA, respectively. In addition to the fact that the cell-free system from uninfected cells synthesized late proteins, no translational discrimination against early mRNA in favor of late mRNA was detected in the cell-free systems from T7 infected cells taken late in T7 infection. Therefore, the functional decay of T7 early mRNA occurring after the shut-off of its synthesis, as reported by us previously, seems sufficient to explain the preferential synthesis of late proteins late in T7 infection in the presence of both chemically stable early mRNA and newly-synthesized late mRNA without necessitating translational control.

Macromolecular synthesis in T7 infected F′ cells

The abortive development of bacteriophage T7 in Escherichia coli cells carrying F′ factors has previously been attributed to lack of virus-directed modification of ribosomes in such cells. However, we find that 6–8 min after T7 infection of male bacteria there is an overall cessation of macromolecular synthesis, resulting in reduced production of DNA, late messenger RNA, and both early and late T7 proteins. Processing of an early viral protein has been detected in F − hosts; this processing is incomplete in F′ infection. The overall paralysis of macromolecular metabolism is correlated in time with a permeability change resulting in loss of the entire acid-soluble pool of phosphorus-containing compounds from the cells ( Britton and Haselkorn, 1975). Thus T7 development in F′ cells appears to be arrested at a particular time without regard to the developmental stage reached by T7 at that time.

The mechanism of inhibition of reovirus replication by interferon

Experiments were carried out to identify the reovirus function that is the primary target for inhibition in interferon-treated cells. The two earliest functions, adsorption and conversion of parental virions to subviral particles, were completely resistant to interferon. The transcription of early messenger RNA was slightly sensitive; the transcription of all 10 species of single-stranded RNA was inhibited by 15–25% at 75 PRD 50/ml and by 50–60% at 300 PRD 50/ml interferon. No RNA species of abnormal size were detected in interferon-treated cells. Subviral particles isolated from cells treated with 300 PRD 50/ml interferon transcribed RNA as actively as subviral particles isolated from normal cells. The translation of early messenger RNA was inhibited much more severely, the degree of inhibition for the individual messenger RNA species ranging from 36 to 72% at 75 PRD 50/ml interferon. The most sensitive species was that which codes for polypeptide λ1. Three late functions, namely, the formation of progeny double-stranded RNA, the transcription of late messenger RNA, and the formation of infectious progeny, were all about as sensitive (75–90% inhibition at 75 PRD 50/ml interferon) as the translation of polypeptide λ1. This polypeptide is a constituent of double-stranded RNA-synthesizing structures, without the formation of which no late reovirus functions can be expressed. The results supported the conclusion that the reason why the replication of reovirus is inhibited in cells treated with interferon is that in such cells the translation of early reovirus mRNA, particularly that which codes for polypeptide λ1, is suppressed.

Poly(A) sequences of vaccinia virus messenger RNA: Nature, mode of addition and function during translation in vitro and in vivo

Studies were undertaken of the nature and mode of addition of the poly(A) sequences at the 3′-termini of vaccinia virus messenger RNA, as well as of their function during translation in vivo and in vitro. The median length of poly(A) sequences on messenger RNA synthesized in vivo, both “early” and “late” in infection, and on RNA transcribed by vaccinia virus cores in vitro was the same, namely about 100 residues. Poly(A) on messenger RNA synthesized in vivo was slightly more homogeneous in size than that on RNA transcribed in vitro. No nucleic acid bases other than adenine were present. Base composition analysis of poly(A) liberated by pancreatic and T1 ribonuclease indicated that the average 3′-terminal sequences of vaccinia virus RNA were G(C2 U4 A?)A100 Aoh.When vaccinia virus messenger RNA was hybridized to DNA, the poly(A) sequences remained free, and at least 95% of them could be removed by treatment with pancreatic and/or T1 ribonuclease, with no detectable diminution in size. This was interpreted as evidence that they were added posttranscriptionally, rather than copied from sequences of poly(dT) in the viral genome. In fact, the vaccina virus strain WR genome was found to contain essentially no poly(dT) sequences (less than 0.015% of the virus genome) as judged by poly(A) binding.In the presence of cordycepin, the formation of both early and late vaccinia messenger RNA in infected cells was markedly inhibited (90% inhibition by 50 μg/ml). The residual 10% of messenger RNA that was still formed under these conditions contained poly(A) sequences that were only about one-half their normal length, but gave rise to about one-half of the amount of virus-specified protein that was synthesized in the absence of cordycepin. Halving the length of poly(A) sequences on vaccinia virus messenger RNA therefore appears to have no adverse effects on its ability to be translated in vivo.Not all vaccinia virus messenger RNA molecules possessed poly(A); both early and late messenger RNA synthesized in vivo, as well as RNA transcribed in vitro, contained about 10% of molecules that did not bind to poly(U)-cellulose and contained no poly(A). The poly(A) (−) RNA molecules that were transcribed in vitro had the same size as poly(A) (+) RNA, and possessed the same base sequences as judged by hybridization analysis. Further, they were translated as efficiently as poly(A) (+) RNA molecules in an in vitro Krebs II ascites cell-free protein synthesizing system, yielding the same polypeptides. The presence of poly(A) was therefore not essential for translation in vitro. The pattern of polypeptides that was obtained was complex; this was no doubt due to the fact that, as determined by hybridization analysis, the messenger RNA synthesized by vaccinia virus cores in vitro was transcribed from 50% of the viral genome. This degree of complexity was quantitatively comparable to that of early messenger RNA synthesized in vivo.

Replication of bacteriophage λ DNA dependent on the function of host and viral genes: I. Interaction of red, gam and rec

We have studied the role of the red and gam genes in lambda replication, after infection of wild type and two recombination deficient hosts. Our results show that the rate of phage DNA replication is abnormally low in the absence of red function, in rec + as well as rec − ( A − and A − B −) bacteria. It appears that the virus general recombination proteins play some role in lambda replication that cannot be assumed by the general recombination proteins of its bacterial host. The red − defect in replication results in a decrease in the total amount of intracellular phage DNA. This DNA, nevertheless, seems normal in structure and is matured and packaged with good efficiency. In rec + and recA − hosts infected with gam − mutants, the rate of lambda replication is also low, but in this case, abnormal DNA structures are produced at late times. The gam mutation seems to alter the program of replication such that circular molecules are produced not only at early times, but continuously, throughout the lytic cycle. This, and other facts, suggest that the gam protein is required for the transition from “early” to “late” replication. This requirement for gam function is not observed in recA −B − hosts, in which gam mutants replicate at a normal rate and produce DNA indistinguishable from that made by wild type phage. Thus, the gam requirement seems to involve an interaction of this phage protein with the product of the host's recB gene. Other evidence for such interaction comes from our finding that, in vivo, the gam protein does inhibit presumed action of the host's BC nuclease. In the gam − mutant infections, which are blocked in late replication, absence of a general recombination system seems to create a severe defect in maturation of intracellular phage DNA. This defect, unlike the one affecting λ replication rate, can be alleviated by either the red or rec functions and is correlated with the inability of the mutant phages to make DNA concatemers. Since other late functions (i.e. late messenger RNA production) appear to be normal, we conclude that concatemer formation, via replication or recombination, is an essential step in phage development.

An RNA polymerase mutant of Escherichia coli defective in the T4 viral transcription program

Some rifampicin-resistant mutants of E. coli, which grow normally, poorly support the growth of bacteriophage T4. One such mutant, Rif R-2, has been studied in some detail. The resistance to rifampicin and the effect on T4 development are cotransduced with arg H and the RNA polymerase is rifampicin resistant, which proves that both effects are due to a mutation in RNA polymerase. The phenotype of T4 development in Rif R-2 has been examined. There is a slight delay in the onset of T4 DNA replication, and at most a slight delay in the synthesis of T4 late messenger RNA, but a considerable delay in the synthesis or assembly of T4 tail fibers. This latter defect is probably sufficient to account for the delay in phage production and can be explained either by a relative inability to make the structural gene products of the tail fibers or through a defect in the synthesis of the gene 57 product—a delayed early T4 protein. Infection of Rif R-2 by T4 results in an incomplete cessation of E. coli specific transcription which may be related to the inability to make tail fibers.

Control of late messenger RNA synthesis during λ phage development

In order to study the role of protein and DNA synthesis in the late gene expression of phage λ, the effects of chloramphenicol and thymidine deprivation on λ mRNA synthesis were investigated. After λ phage infection, normal transcription of late genes, as RNA hybridizable with imm 80 h λ DNA, started 8–10 min after infection, and the rate of synthesis continued to increase almost linearly until lysis. Chloramphenicol, added at or before phage infection, virtually inhibited late transcription. When chloramphenicol was added at various stages of the late phase (after onset of late transcription), the increase of late transcription normally observed was slowed down, and afterwards the rate of late transcription decreased gradually. Similar inhibition of late transcription was also observed when DNA synthesis was inhibited by thymidine deprivation. Under the conditions of the present experiments, chloramphenicol permitted considerable synthesis of λ DNA, whereas thymidine deprivation inhibited DNA synthesis to below 5 % of the control. These results seem to suggest that normal continuation of late transcription requires both replication of phage DNA and protein synthesis continuously throughout λ phage development. Such processes relating to the regulatory mechanism of late transcription are discussed.

Role of initiation factor B (F3) in the preferential translation of T4 late messenger RNA in T4 infected E. Coli

Role of initiation factor B (F3) in the preferential translation of T4 late messenger RNA in T4 infected E. Coli

Effect of mutations in the cII and cIII genes of bacteriophage λ on macromolecular synthesis in infected cells

We have investigated the time-course of macromolecular synthesis following infection by cI −, cII − and cIII − mutants of λ in an effort to understand the role of the cII and cIII genes in the establishment of repression and lysogeny. cII − and cIII − mutants of λ begin to synthesize the “late” proteins tail antigen and lysozyme substantially before a cI − mutant; the shift to late messenger RNA production from the head and tail genes also occurs earlier after cII − or cIII − infection. In contrast, no appreciable difference among cI −, cII −, or cIII − mutants was found in synthesis of the “early” protein λ-exonuclease or in the rate of total or λ-specific DNA synthesis. These results suggest that the λ cII and cIII gene products function to delay the lytic response by inhibiting, directly or indirectly, the production of messenger RNA from the late λ genes. When lysozyme synthesis was studied after infection by cI − cII − and cI − cIII − double mutants, results were obtained which were similar to those found in the case of single cII − and cIII − mutants. The results with the double mutants suggest that the cII and cIII gene products may also participate in the regulation of cI represser synthesis or activity.

Function of T4 gene 55: II. RNA synthesis by temperature-sensitive gene 55 mutants

T4 protein-dependent late messenger RNA and DNA synthesizing capacities accumulate simultaneously between 7 and 11 minutes after infection at 30 °C. While actual DNA replication starts at six to eight minutes after infection, late messenger RNA is first observed at 11 to 12 minutes after infection (i.e. 4 to 5 min after the first appearance of the proteins required for the early-late switch). This lag probably reflects the time intervening between the initiation of DNA synthesis and the formation of the first late transcription competent template. T4 gene 55 product exerts a positive, continuous and probably direct control on late transcription. Shift-up to 43 °C of cells infected by a gene 55 temperature-sensitive mutant ( ts553) leads to an arrest in late transcription within three minutes. Under the same conditions, lysozyme synthesis drops by an order of magnitude in about 15 minutes. If cells infected by ts553 at 43 °C are shifted to 30 °C, late transcription resumes immediately even if protein synthesis is inhibited by the addition of chloramphenicol.

RNA metabolism in T4-infected Escherichia coli

Discrete species of 14C-labeled messenger RNA from T4-infected Escherichia coli can be detected as bands on polyacrylamide gels after electrophoresis and autoradiography. Experiments are presented which demonstrate that the transcriptional patterns change during the course of infection and that protein synthesis inhibition at appropriate times of infection prevents certain transcriptional transitions. It is shown that the synthesis of stable host RNA (23 s, 16 s, 5 s and transfer RNA) ceases after two minutes of infection at 25 °C but that the addition of chloramphenicol to the culture before infection prevents this shut off. “Early” phage RNA which is synthesized before the onset of DNA synthesis can be divided into two subclasses by virtue of the fact that only one class is synthesized in chloramphenicol pretreated cultures. Also, chloramphenicol prevents the synthesis of late phage RNA even if added at a time when it allows a significant amount of phage DNA to be synthesized. This indicates that phage DNA synthesis is a necessary but not a sufficient requirement for transcription of the late phage genes. Analysis of the RNA synthesized by cultures infected with various phage mutants demonstrates that phage DNA synthesis and the functioning of genes 55 and 33 are required for the synthesis of late messenger RNA which is synthesized after 20 minutes of infection at 25 °C. The striking similarity of the RNA patterns from cultures infected with amber mutants in genes 33 and 55 corroborates their classification in the same class of maturation defectiveness. It is shown that though almost all of the RNA labeled during phage infection is unstable, several RNA species in the molecular weight range 20,000 to 50,000 are stable and accumulate in large amounts during infection. The results are discussed in relation to our current understanding of the mechanisms controlling RNA and protein synthesis in phage-infected cells.

Studies on a temperature-sensitive mutant of vaccinia virus strain WR

A number of temperature-sensitive mutants of vaccinia virus strain WR have been isolated. Some are more heat labile than wild-type virus and are probably capsid protein mutants. One mutant of a different type, ts 20.165, was investigated in some detail. This mutant has a delayed and decreased rate of DNA replication, as well as a diminished rate of early messenger RNA transcription at the nonpermissive temperature (39°). However, both of these defects, and especially the latter, also operate at the permissive temperature (31°), at which the viral yield is as high as that of wild-type virus. Late functions, such as the transcription of late messenger RNA and switch-off of the synthesis of DNA polymerase, are normal. Temperature-shift experiments confirmed that ts 20.165 is an early function mutant. ts 20.165 virions have associated with them less than one-half of the amount of RNA polymerase associated with wild-type virions. Further, the DNA polymerase coded by the mutant is about twice as heat labile as the enzyme coded by wild-type virus. ts 20.165 virus particles are formed at 39°. These particles have only about 3% of the specific infectivity of particles formed at 31°. They contain, as far as one can tell, a normal amount of DNA and a normal capsid protein complement, and are normal in appearance. However, the yield at 39° has a higher frequency of revertants, on a PFU basis, than the yield at 31°. ts 20.165 recombines with high efficiency with two other ts mutants of vaccinia WR. Complementation is weak, but detectable. It is concluded that ts 20.165 is an early function mutant. The possible nature of its defect is discussed.

Control of RNA synthesis during phage development: Isolation and properties of a λ-∅81 hybrid phage

Based on the finding that very little homology exists between λ and ∅81 or ∅80 phage at the level of RNA, early and late messenger RNA have been conveniently assayed by using hybrid phages, Hyb10 and imm 80 h λ . Hyb10 phage carries most of the genes of ∅81 except for “early genes” that are of λ origin. In agreement with this genetic constitution, exonuclease found in Hyb10-infected cells is of λ type, whereas endolysin is of ∅81 type with respect to the several properties examined. Messenger RNA formed at various stages of infection with λ vir phage hybridized equally well with denatured DNA of Hyb10 phage. In contrast, the fraction of RNA hybridizable with DNA of imm 80 h λ phage was extremely low in the first 10 minutes and increased markedly later in infection. These results show that early messenger RNA synthesis continues through the late phase at more or less constant rate, whereas extensive synthesis of late messenger RNA begins only after some lag. Experiments with Hyb10-infected cells gave results that are consistent with the thesis that a receptor site(s) for the immunity substance is located within the early region of the phage genome, and that repression takes place at the level of messenger RNA synthesis.

In vitro synthesis of T4 late messenger RNA.

In vitro synthesis of T4 late messenger RNA.

Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

The vaccinia virus multiplication cycle can be divided into two portions: the early portion, which precedes viral DNA replication and lasts for about one-and-a-half hours; and the late portion which follows until maturation of progeny is complete. Vaccinia messenger RNA transcribed during these two periods, that is, early and late messenger RNA, has been studied by means of hybridization and density gradient sedimentation. The following results were obtained: 1. (1) Early vaccinia messenger RNA is distinctly smaller than late messenger RNA (10 to 12 s against 16 to 20 s). Late messenger RNA contains nucleotide sequences not present in early messenger RNA; however, all the sequences transcribed early are also transcribed late. If late messenger RNA is transcribed from the entire viral genome, one-half to two-thirds is transcribed early. 2. (2) Messenger RNA transcribed in the presence of cytosine arabinoside, which completely inhibits viral DNA replication, appears to be identical to early messenger RNA. The cytosine arabinoside RNA transcribed in HeLa and L cells contains the same base sequences. 3. (3) The pattern of transcription of early and late vaccinia messenger RNA in HeLa and L cells is quite different. In HeLa cells the total amount of late messenger RNA synthesized greatly exceeds the amount of early; in L cells the position is reversed. 4. (4) The effect of varying the multiplicity of infection on the pattern of early and late messenger RNA transcription has been studied. The rate of early mRNA synthesis is proportional to the multiplicity over a certain limited range. Once the maximum rate of mRNA transcription has been reached, which occurs the earlier the higher the multiplicity, the rate of transcription of messenger RNA falls to a steady low level equal to no more than 5% of the maximum value; this rate of transcription then persists until at least 19 hours after infection. 5. (5) The large messenger RNA molecules transcribed late contain nucleotide sequences also present in small early messenger RNA molecules. Some small messenger RNA molecules are also transcribed late: these contain at least some sequences characteristic of late messenger RNA. 6. (6) At five hours after infection the messenger RNA molecules present in polyribosomes contain all the sequences characteristic of early messenger RNA molecules. By eight hours after infection messenger RNA in polyribosomes is very significantly depleted with respect to sequences characteristic of early messenger RNA. 7. (7) The stability of early and late vaccinia messenger RNA in the presence of actinomycin D has been studied in HeLa and L cells. Early vaccinia messenger RNA is very stable in HeLa cells. Those late messenger RNA molecules which contain sequences characteristic of late messenger RNA are mostly unstable (half-life less than one hour); but those containing sequences characteristic of early messenger RNA are as stable as early messenger RNA itself. In L cells early and late vaccinia messenger RNA are equally stable (half-life two to three hours). mRNA is considerably more stable in extracts of HeLa cells than of L cells; and early mRNA is somewhat more stable in extracts of HeLa cells than is late mRNA. These results are discussed in relation to the pattern of macromolecular biosynthesis during the vaccinia virus multiplication cycle.

Presence of T4 “early” messenger RNA on polysomes late in infection

Hybridization of T4 pulse-labeled RNA to denatured DNA was used to determine whether “early” messenger RNA is found in association with polyribosomes at late times after infection. Results indicate that almost all classes of “early” messenger RNA are attached to polysomes late in infection, and that the distribution of “early” and late messenger RNA on late polysomes is similar.