Transcription of the bacteriophage T4 template. Detailed comparison of in vitro and in vivo transcripts

Biochemistry ◽  
1970 ◽  
Vol 9 (6) ◽  
pp. 1300-1309 ◽  
Author(s):  
Edward N. Brody ◽  
E. Peter Geiduschek
Keyword(s):  
Bacteriophage T4 ◽  
Detailed Comparison

scholarly journals Mutational analysis of the mRNA operator for T4 DNA polymerase.

Genetics ◽  
1991 ◽  
Vol 128 (2) ◽  
pp. 203-213 ◽  
Author(s):  
M D Andrake ◽  
J D Karam
Keyword(s):  
Dna Polymerase ◽  
Mutational Analysis ◽  
Bacteriophage T4 ◽  
Spatial Arrangement ◽  
Loop Sequence ◽  
Shine Dalgarno Sequence ◽  
In Vitro Effects ◽  
Rna Hairpin

Abstract Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

scholarly journals Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations

2001 ◽  
Vol 183 (7) ◽  
pp. 2289-2297 ◽  
Author(s):  
Marco P. Cicero ◽  
Meghan M. Sharp ◽  
Carol A. Gross ◽  
Kenneth N. Kreuzer
Keyword(s):  
Rna Polymerase ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Positive Control ◽  
Plaque Morphology ◽  
Mutant Proteins ◽  
E Coli

ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. AmotA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. colipromoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

T4 Replication Accessory Proteins Visualized as Complexes with DNA by Cryoelectron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 264-265
Author(s):  
Edward P. Gogol ◽  
Mark C. Young ◽  
William L. Kubasek ◽  
Thale C. Jarvis ◽  
Peter H. von Hippel
Keyword(s):  
Bacteriophage T4 ◽  
Dna Binding Protein ◽  
Cryoelectron Microscopy ◽  
Accessory Proteins ◽  
Replication Process ◽  
Atp Analogues ◽  
Replication Activity ◽  
Association State

Replication of bacteriophage T4 is accomplished by a set of proteins encoded by the T4 genome.Central to replication is the DNA polymerase, the product of gene 43 (g43p); while it contains the intrinsic functions of template-directed DNA synthesis, it requires a set of accessory proteins as a minimal system to function with a rate, fidelity and processivity comparable to those obtained in vivo. These accessory proteins are g44p, g45p and g62p; g32p, a single-stranded DNA binding protein, also assists in the replication process, both in vitro and in vivo. In the absence of DNA, g44p and g62p form a 4:1 complex which has a DNA-dependent ATPase activity; g45p is a trimer in solution. Their association state when bound to DNA is however unknown, as is the mechanism by which they dramatically improve the replication activity of g43p.We have examined the complex of the three replication accessory proteins and g32p as a complex with DNA by cryoelectron microscopy, avoiding stains or fixatives which may disrupt the labile structures formed. Under conditions which promote the polymerase-enhancing activity of this four-protein system, distinct "hash mark" structures are visible along strands of replicative form M13 DNA (figure 1). These structures are formed only in the presence of all four proteins and ATP; absence of any one of the proteins or ATP, or substitution of non-hydrolyzable ATP analogues, fails to yield any of the hash marks.

scholarly journals In Vivo Bypass of Chaperone by Extended Coiled-Coil Motif in T4 Tail Fiber

2004 ◽  
Vol 186 (24) ◽  
pp. 8363-8369 ◽  
Author(s):  
Yun Qu ◽  
Paul Hyman ◽  
Timothy Harrah ◽  
Edward Goldberg
Keyword(s):  
Bacteriophage T4 ◽  
Coiled Coil ◽  
Critical Factor ◽  
Coiled Coils ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Type Gene ◽  
In Vivo Screen

ABSTRACT The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35. Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30°C but not at 42°C was found in gene 37 (R. J. Bishop and W. B. Wood, Virology 72:244-254, 1976). Sequencing of the temperature-sensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S. Hashemolhosseini et al., J. Mol. Biol. 241:524-533, 1994). We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation. To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil. Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads. While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50°C. This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37. The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism. The increased melting temperature should be useful for “clamps” to initiate the folding of trimeric β-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils.

scholarly journals Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
Author(s):  
Umender K. Sharma ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Sigma Factors ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

scholarly journals Bacteriophage T4 Self-Assembly: In Vitro Reconstitution of Recombinant gp2 into Infectious Phage

2000 ◽  
Vol 182 (3) ◽  
pp. 672-679 ◽  
Author(s):  
G. R. Wang ◽  
A. Vianelli ◽  
E. B. Goldberg
Keyword(s):  
Self Assembly ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
Biologically Active ◽  
Phage Infection ◽  
Direct Role ◽  
In Vitro Reconstitution ◽  
Phage Dna

ABSTRACT T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in therecBCD + host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2+ phage infection (H+) with tails from a gp2+ or gp2− phage infection (T+ or T−), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H−) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV+ cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2+ phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.

DNA Polymerase Fidelity: From Genetics Toward a Biochemical Understanding

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1475-1482
Author(s):  
Myron F Goodman ◽  
D Kuchnir Fygenson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Theoretical Models ◽  
Model Systems ◽  
Polymerase Fidelity ◽  
Base Selection ◽  
Physical Biochemistry

Abstract This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful—first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered. For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.

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