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1.
J Virol ; 75(23): 11263-74, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11689606

ABSTRACT

A Moloney murine leukemia virus-based single-replication-cycle assay was developed to study the effects of limiting the extent of template and primer strand complementarity on recombinogenic template switching. This system mimicked forced copy choice recombination in which nascent DNA transfers from the end of a donor template to an acceptor position on the other copackaged RNA. When acceptor target regions with different extents of complementarity to the transferring DNA were tested, efficient recombination occurred with as few as 14 complementary nucleotides. The frequencies of correct targeting, transfer-associated errors, mismatch extension, and transfer before reaching the end of the donor template were determined. All four molecular events occurred, with their proportions varying depending on the nature of acceptor/transferring DNA complementarity. When complementarity was severely limited, recombination was inefficient and most products resulted from aberrant second-strand transfer rather than from forced template switching between RNAs. Other classes of reverse transcription products, including some that resulted from template switching between virus and host sequences, were also observed when homology between the acceptor and donor was limited.


Subject(s)
Moloney murine leukemia virus/physiology , Recombination, Genetic , Sequence Homology, Nucleic Acid , Virus Replication , 3T3 Cells , Animals , Base Pair Mismatch , Base Sequence , DNA, Viral/genetics , Mice , Molecular Sequence Data , Moloney murine leukemia virus/genetics , RNA, Viral/genetics , Templates, Genetic , Transcription, Genetic
2.
Virology ; 286(2): 475-82, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11485415

ABSTRACT

Retroviral genetic rearrangements can result from reverse transcriptase template switching. Most published data suggest that errors such as base misincorporation occur at similar frequencies for HIV-1 and for simple retroviruses such as spleen necrosis virus (SNV) and murine leukemia virus (MuLV). However, previous reports have suggested that template switch-mediated recombination is much more frequent for HIV-1 than for simple retroviruses. In this report, direct repeat deletion vectors similar to those previously used for measuring template switching events for SNV and MuLV were developed for HIV-1. Forward mutation rates and the frequency of template switching during a single cycle of HIV-1 replication were determined. The frequency of HIV-1-mediated repeat deletion was measured for three separate internal repeats in lacZ and was compared to rates observed with identical repeats for MuLV. The results indicated that the error rate and the frequency of repeat deletion of HIV-1 were similar to those of MuLV.


Subject(s)
Gene Deletion , Genetic Vectors , HIV-1/genetics , Recombination, Genetic , Repetitive Sequences, Nucleic Acid/genetics , Transcription, Genetic , Cell Line , DNA, Viral/metabolism , HIV-1/metabolism , Humans , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/metabolism , Templates, Genetic , Virus Replication
3.
J Virol ; 74(22): 10293-303, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11044073

ABSTRACT

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.


Subject(s)
DNA, Viral/genetics , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/physiology , Purines/chemistry , Virus Replication , Base Sequence , DNA, Viral/chemistry , Genetic Variation , Mutation , Proviruses/genetics , Sequence Deletion
4.
J Virol ; 74(20): 9629-36, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11000235

ABSTRACT

Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functional lacZ gene generation from vectors in which lacZ was disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.


Subject(s)
Moloney murine leukemia virus/genetics , RNA-Directed DNA Polymerase/chemistry , Transcription, Genetic , DNA/biosynthesis , Mutation , Tandem Repeat Sequences , Virus Replication
5.
J Virol ; 74(6): 2694-702, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10684285

ABSTRACT

It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral "headful" was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19. 2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.


Subject(s)
Moloney murine leukemia virus/genetics , Virus Replication/genetics , 3T3 Cells , Animals , DNA, Viral , Genetic Vectors , Genome, Viral , Mice , Moloney murine leukemia virus/physiology , Proviruses/genetics , RNA, Viral/biosynthesis , Templates, Genetic , Virus Assembly/physiology
6.
J Virol ; 73(10): 8441-7, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10482596

ABSTRACT

During retroviral DNA synthesis reverse transcriptase frequently performs nonrequired template switches that can lead to genetic rearrangements or recombination. It has been postulated that template switching occurs after pauses in the action of reverse transcriptase. Hence factors which affect pausing, such as polymerization rate, may affect the frequency of template switching. To address the hypothesis that increasing the time required to complete reverse transcription increases the frequency of template switching, we established conditions that lengthened the time required to complete a single round of intracellular Moloney murine leukemia virus reverse transcription approximately threefold. Under these conditions, which resulted from intracellular nucleotide pool imbalances generated with hydroxyurea, we examined template switching frequency using a lacZ-based tandem repeat deletion assay. We observed that the frequency of deletion during reverse transcription in hydroxyurea-treated cells was approximately threefold higher than that in untreated control cells. These findings suggest that rates of retroviral recombination may vary when the intracellular environment is altered.


Subject(s)
DNA, Viral/genetics , Leukemia Virus, Murine/physiology , Transcription, Genetic/physiology , 3T3 Cells , Animals , DNA, Viral/biosynthesis , Mice , RNA-Directed DNA Polymerase/genetics , Recombination, Genetic , Sequence Deletion , Templates, Genetic , Virus Replication
7.
J Virol ; 73(2): 948-57, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9882295

ABSTRACT

A conserved purine-rich motif located near the 3' end of retroviral genomes is involved in the initiation of plus-strand DNA synthesis. We mutated sequences both within and flanking the Moloney murine leukemia virus polypurine tract (PPT) and determined the effects of these alterations on viral DNA synthesis and replication. Our results demonstrated that both changes in highly conserved PPT positions and a mutation that left only the cleavage-proximal half of the PPT intact led to delayed replication and reduced the colony-forming titer of replication defective retroviral vectors. A mutation that altered the cleavage proximal half of the PPT and certain 3' untranslated region mutations upstream of the PPT were incompatible with or severely impaired viral replication. To distinguish defects in plus-strand priming from other replication defects and to assess the relative use of mutant and wild-type PPTs, we examined plus-strand priming from an ectopic, secondary PPT inserted in U3. The results demonstrated that the analyzed mutations within the PPT primarily affected plus-strand priming whereas mutations upstream of the PPT appeared to affect both plus-strand priming and other stages of viral replication.


Subject(s)
3' Untranslated Regions , Moloney murine leukemia virus/genetics , Mutation , Virus Replication , 3T3 Cells , Animals , Base Sequence , Cell Line , DNA, Viral/biosynthesis , Genetic Vectors , Humans , Mice , Molecular Sequence Data , Moloney murine leukemia virus/physiology , Proviruses
8.
J Mol Biol ; 281(1): 1-15, 1998 Aug 07.
Article in English | MEDLINE | ID: mdl-9680471

ABSTRACT

Template switching is required during normal retroviral DNA synthesis and is involved in retroviral genetic recombination. The first strong stop template switch during Moloney murine leukemia virus reverse transcription was studied to examine how template switch acceptor templates are selected. Retroviral vectors with specific alterations in their template switch acceptor regions were constructed, and DNA products templated by these vectors during a single replication cycle were analyzed. The results indicated that maximizing complementarity between the primer strand 3' end and the acceptor template was not the most significant factor in determining a strong stop template switch site. Instead, preferential transfer to the U3/R junction was observed, with as little as one contiguous base-pair of complementarity between the primer terminus and the template strand sufficient to direct template switching to the U3/R junction. These findings suggest that, in contrast to prevailing dogma, a base-pairing-independent mechanism functions in the specific guidance of retroviral strong stop template switch to the U3/R junction. Certain template alterations 3' of the template switch site were at least as disruptive to acceptor template use as was primer-terminal mismatch, suggesting that template structure or primer strand-internal sequences are important determinants of acceptor template selection. We discuss the implications of these findings for the mechanisms of retroviral DNA synthesis and homologous recombination.


Subject(s)
Moloney murine leukemia virus/genetics , 3T3 Cells , Animals , Base Sequence , DNA, Viral/biosynthesis , DNA, Viral/genetics , Genetic Complementation Test , Genetic Vectors , Mice , Molecular Sequence Data , Moloney murine leukemia virus/physiology , Mutation , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , Recombination, Genetic , Transcription, Genetic , Virus Replication
9.
EMBO J ; 16(4): 856-65, 1997 Feb 17.
Article in English | MEDLINE | ID: mdl-9049314

ABSTRACT

Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus-based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfer nearly always occurred precisely at the 5' end of genomic RNA. This finding allowed us to use first strand transfer to study the classes of errors that reverse transcriptase can and/or does make when it switches templates at a defined position during viral DNA synthesis. We found that errors occurred at the site of first strand transfer approximately 1000-fold more frequently than reported average reverse transcriptase error rates for template-internal positions. We then analyzed replication products of specialized vectors that were designed to test possible origins for the switch-associated errors. Our results suggest that at least some errors arose via non-templated nucleotide addition followed by mismatch extension at the point of strand transfer. We discuss the significance of our findings as they relate to the possible contribution that template switch-associated errors may make to retroviral mutation rates.


Subject(s)
Moloney murine leukemia virus/genetics , RNA, Viral/genetics , RNA-Directed DNA Polymerase/metabolism , Transcription, Genetic/genetics , Animals , DNA, Viral/biosynthesis , Genetic Vectors/genetics , Mice , Moloney murine leukemia virus/enzymology , Point Mutation
10.
Structure ; 3(9): 879-92, 1995 Sep 15.
Article in English | MEDLINE | ID: mdl-8535782

ABSTRACT

BACKGROUND: Reverse transcriptase (RT) converts the single-stranded RNA genome of a retrovirus into a double-stranded DNA copy for integration into the host genome. This process requires ribonuclease H as well as RNA- and DNA-directed DNA polymerase activities. Although the overall organization of HIV-1 RT is known from previously reported crystal structures, no structure of a complex including a metal ion, which is essential for its catalytic activity, has been reported. RESULTS: Here we describe the structures at 1.8 Angstrum resolution of a catalytically active fragment of RT from Moloney murine leukemia virus (MMLV) and at 2.6 Angstrum of a complex of this fragment with Mn2+ coordinated in the polymerase active site. On the basis of similarities with HIV-1 RT and rat DNA polymerase beta, we have modeled template/primer and deoxyribonucleoside 5'-triphosphate substrates into the MMLV RT structure. CONCLUSIONS: Our model, in the context of the disposition of evolutionarily conserved residues seen here at high resolution, provides new insights into the mechanisms of catalysis, fidelity, processivity and discrimination between deoxyribose and ribose nucleotides.


Subject(s)
Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/metabolism , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , DNA Primers/chemistry , DNA Primers/metabolism , Deoxyribonucleotides/chemistry , Deoxyribonucleotides/metabolism , HIV-1/enzymology , Metals/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Ribonucleoproteins/chemistry , Sequence Alignment
11.
Science ; 267(5194): 96-9, 1995 Jan 06.
Article in English | MEDLINE | ID: mdl-7528942

ABSTRACT

Replication complexes that contained either murine leukemia virus reverse transcriptase (MLV RT) or a variant reverse transcriptase without a ribonuclease (RNase) H domain (delta RH MLV RT) were visualized by enzymatic footprinting. Wild-type MLV RT protected template nucleotides +6 to -27, and primer nucleotides -1 to -26 of primers that had first been extended by one or four nucleotides. Although it catalyzed DNA synthesis, delta RH MLV RT stably bound template-primer only under conditions of reduced ionic strength and protected the duplex portion only as far as position -15. Despite altered hydrolysis profiles, both enzymes covered primarily the template-primer duplex, contradicting recent predictions based on the structure of rat DNA polymerase beta.


Subject(s)
Leukemia Virus, Murine/enzymology , RNA-Directed DNA Polymerase/metabolism , DNA/biosynthesis , DNA Primers/metabolism , Deoxyribonuclease I/metabolism , HIV Reverse Transcriptase , Hydrolysis , RNA-Directed DNA Polymerase/chemistry , Recombinant Proteins/metabolism , Ribonuclease H/metabolism , Templates, Genetic
13.
EMBO J ; 12(11): 4433-8, 1993 Nov.
Article in English | MEDLINE | ID: mdl-7693456

ABSTRACT

Retroviral DNA synthesis requires both the DNA polymerase and the RNaseH activities of reverse transcriptase (RT). To test whether two defective RTs--one carrying a mutation in the RNaseH domain and the other with a mutation in DNA polymerase--could work together to complete viral DNA synthesis, we generated phenotypically mixed virions of Moloney murine leukemia virus (M-MuLV) that contained two kinds of mutant RTs. One RNaseH catalytic site mutant complemented both tested DNA polymerase mutants and small amounts of intact viral DNA were generated. This demonstrates that retroviral DNA synthesis can be completed--albeit inefficiently--when DNA polymerase and RNaseH activities are provided by separate RT molecules. Other RNaseH mutants failed to complement, suggesting that some aspects of the RNaseH domain are essential to RT's DNA polymerase function. Phenotypically mixed virions were also used to demonstrate that RT and integrase (IN) can be provided by separate polyprotein precursors and complete the early stages of retroviral replication.


Subject(s)
Genetic Complementation Test , Moloney murine leukemia virus/genetics , Mutation , RNA-Directed DNA Polymerase/genetics , 3T3 Cells , Animals , DNA Nucleotidyltransferases/genetics , DNA-Directed DNA Polymerase/metabolism , Integrases , Mice , Moloney murine leukemia virus/enzymology , Moloney murine leukemia virus/pathogenicity , Phenotype , Proviruses/enzymology , Proviruses/genetics , RNA-Directed DNA Polymerase/metabolism , Rats , Retroviridae Proteins/genetics , Ribonuclease H/metabolism , Virion/enzymology , Virion/genetics , Virulence , Virus Integration
14.
Proc Natl Acad Sci U S A ; 90(4): 1276-80, 1993 Feb 15.
Article in English | MEDLINE | ID: mdl-7679498

ABSTRACT

The active sites for the polymerase and nuclease activities of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) reside in separate domains of a single polypeptide. We have studied the effects of RNase H domain mutations on DNA polymerase activity. These mutant RTs displayed decreased processivity of DNA synthesis. We also compared complexes formed between primer-templates and mutant and wild-type reverse transcriptase (RT). Although M-MuLV RT is monomeric in solution, two molecules of RT bound DNA cooperatively, suggesting that M-MuLV RT binds primer-template as a dimer. Some mutant RTs with decreased processivity failed to form the putative dimer.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Moloney murine leukemia virus/enzymology , Moloney murine leukemia virus/genetics , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Base Sequence , Binding Sites , Cloning, Molecular , DNA, Viral/genetics , DNA, Viral/metabolism , DNA-Directed DNA Polymerase/genetics , Genes, pol , Macromolecular Substances , Molecular Sequence Data , Mutagenesis , Oligodeoxyribonucleotides , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Ribonuclease H/genetics , Templates, Genetic
15.
J Virol ; 66(2): 615-22, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1370551

ABSTRACT

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.


Subject(s)
Chromosome Deletion , Escherichia coli/enzymology , Moloney murine leukemia virus/physiology , RNA-Directed DNA Polymerase/genetics , Ribonuclease H/genetics , Virus Replication , 3T3 Cells , Animals , Base Sequence , Cloning, Molecular , DNA, Viral/genetics , DNA, Viral/isolation & purification , Escherichia coli/genetics , Mice , Models, Structural , Molecular Sequence Data , Molecular Weight , Moloney murine leukemia virus/genetics , Oligodeoxyribonucleotides , Protein Conformation , RNA-Directed DNA Polymerase/metabolism , Translocation, Genetic , Virion/genetics , Virion/physiology
16.
J Virol ; 65(8): 4387-97, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1712862

ABSTRACT

The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus-strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer-binding sites near the 5' ends of VL30 RNAs.


Subject(s)
DNA, Viral/genetics , Endoribonucleases/genetics , Moloney murine leukemia virus/genetics , Transcription, Genetic , Animals , Blotting, Southern , Cell Line, Transformed , DNA Replication , DNA, Viral/analysis , DNA, Viral/biosynthesis , Endoribonucleases/metabolism , Moloney murine leukemia virus/enzymology , Moloney murine leukemia virus/physiology , Mutagenesis , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H , Viral Proteins/analysis , Viral Proteins/genetics , Virion/enzymology , Virion/genetics , Virus Replication
17.
Biochemistry ; 28(19): 7781-8, 1989 Sep 19.
Article in English | MEDLINE | ID: mdl-2482069

ABSTRACT

The sigma subunit of bacterial RNA polymerase is required for the specific initiation of transcription at promoter sites. However, sigma is released from the transcription complex shortly after transcription is initiated, and elongation proceeds in the absence of sigma. In order to study the position of sigma release, we have developed a method to quantify the photoaffinity labeling produced by an aryl azide positioned at the leading (5'-) end of nascent RNA, as a function of the transcript length [Stackhouse, T.M., & Meares, C.F. (1988) Biochemistry 27, 3038-3045]. Here we compare photoaffinity labeling of transcription complexes containing three natural bacteriophage promoters (lambda PR, lambda PL, and T7 A1) and two recombinant constructs, A1/PR (T7 A1 promoter with the lambda PR transcribed region) and PR/A1 (lambda PR promoter with the T7 A1 transcribed region). Significant photoaffinity labeling of the sigma subunit was observed only on the templates containing the lambda PR promoter region, regardless of the sequence of the transcribed region. These results indicate the molecular interactions responsible for the position of sigma release from the transcription complex mainly involve the nucleotide sequence of the promoter region--rather than the transcribed region--of the DNA template. Further studies on transcription complexes containing the A1/PR and the PR/A1 templates were performed, using polyclonal antibodies against the holoenzyme or against the sigma subunit. These experiments corroborate the promoter dependence of sigma release. They also show a correlation between the release of sigma and stable binding of the transcript by the transcription complex.


Subject(s)
DNA-Directed RNA Polymerases/genetics , Escherichia coli/enzymology , Promoter Regions, Genetic , Sigma Factor/genetics , Transcription Factors/genetics , Affinity Labels , Base Sequence , DNA, Bacterial/genetics , Escherichia coli/genetics , Models, Genetic , Molecular Sequence Data , Precipitin Tests , RNA/genetics , Templates, Genetic , Transcription, Genetic
18.
Biochemistry ; 28(12): 5210-8, 1989 Jun 13.
Article in English | MEDLINE | ID: mdl-2669971

ABSTRACT

Bacteriophages T3 and T7 contain homologous terminators for Escherichia coli RNA polymerase that restrict early phage transcription to the leftmost 20% of the linear phage genomes. These two terminators serve equally well as p-independent terminators in vivo, but their in vitro efficiencies and sensitivity to salt and nucleotide concentrations differ dramatically. Sequence analysis shows that the T7 and T3 terminators differ at only two sites in the region normally accepted as defining terminator function. In order to determine which structural features of these two terminators are responsible for their functional differences, a series of hybrid terminators were constructed in which structural features of the two terminators were systematically interchanged. Transcription of hybrid terminator templates revealed that sequences downstream of the termination release sites are responsible for the differences in efficiency of in vitro termination. These sequences also determine the sensitivity of these terminators to elevated salt concentrations and to alterations of substrate concentrations. Alteration of the sequences in the region between three and seven nucleotides downstream of the final T7Te release site is sufficient to reduce termination efficiency to that of T3Te, and point mutations in this region yield terminators with intermediate efficiency. Hence, the determinants of p-independent terminator efficiency in vitro must include elements of the transcription complex other than the structure of the 3' end of the transcript. The termination differences between T7Te, T3Te, and their hybrid derivatives are overcome in vivo; all of these sites become very efficient. This finding further supports the hypothesis that protein factors or other cellular features enhance the efficiency and specificity of p-independent terminators in vivo.


Subject(s)
Genes, Regulator , T-Phages/genetics , Terminator Regions, Genetic , Transcription, Genetic , DNA/analysis , DNA-Directed RNA Polymerases/genetics , Deoxyribonucleases , Escherichia coli/enzymology , Escherichia coli/genetics , Hydrolysis , In Vitro Techniques , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Hybridization , Peptide Fragments/analysis , RNA, Messenger/analysis , Sequence Homology, Nucleic Acid , Templates, Genetic
19.
J Mol Biol ; 205(2): 315-30, 1989 Jan 20.
Article in English | MEDLINE | ID: mdl-2467003

ABSTRACT

The efficiency of transcription termination at certain well-defined prokaryotic rho-independent terminators depends on the promoter unit from which transcription is initiated. Some promoter units allow substantial readthrough of strong termination signals, a phenomenon we term "factor-independent antitermination". This observation is not easily explained by current models for prokaryotic terminator function that consider the terminator to be a "cassette" involving only sequences and RNA transcript structures in the immediate region of the terminator or directly upstream. When transcription is carried out in vitro employing only purified Escherichia coli RNA polymerase, up to 20 times as many RNA polymerase molecules pass through a particular terminator when transcription is initiated from the E. coli tac promoter unit, as compared to transcription initiated from the T7A1 or rrnB P2 promoters. This effect cannot be attributed to antitermination factors separate from the core RNA polymerase. Similar differences in termination efficiency are found for the same promoters in vivo. These termination differences are affected by sequences just downstream from the start site for transcription, including those in the +25 region of the nascent transcript. These early transcribed sequences can confer factor-independent antitermination onto a heterologous promoter, but only when the sequences are precisely positioned relative to the start site for transcription. We have considered several possible models to explain how early transcribed sequences might affect termination, including those in which the 5' end of the transcript interacts with either the terminator RNA or the polymerase. We favor an alternative model in which these sequences interact with the core RNA polymerase to convert the enzyme from a termination-proficient state (T-state) to a conformation resistant to termination (R-state). Such enzyme conformations may be an important component of factor-dependent antitermination systems.


Subject(s)
Genes, Regulator , Promoter Regions, Genetic , Terminator Regions, Genetic , Transcription, Genetic , Base Sequence , DNA-Directed RNA Polymerases/genetics , Escherichia coli , Models, Genetic , Molecular Sequence Data , Plasmids , RNA, Bacterial/genetics
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