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The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase
Nudler E; Mustaev A; Lukhtanov E; Goldfarb A
An 8-9 bp RNA-DNA hybrid in the transcription elongation complex is essential for keeping the RNA 3' terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 3' segment of RNA can be removed through transcript cleavage. At certain sites, cycles of unwinding-rewinding of the hybrid are coupled to reverse-forward sliding of the transcription elongation complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inch-worming). Thus, the 3'-proximal RNA-DNA hybrid plays the dual role of keeping the active site in register with the template and sensing the helix-destabilizing mismatches in RNA, launching correction through backtracking and cleavage
PMID: 9094712
ISSN: 0092-8674
CID: 17432
Transcription processivity: protein-DNA interactions holding together the elongation complex
Nudler E; Avetissova E; Markovtsov V; Goldfarb A
The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the beta' subunit and the R interaction, to the COOH-terminal catalytic domain of the beta subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity
PMID: 8662499
ISSN: 0036-8075
CID: 17433
Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase
Kashlev M; Nudler E; Severinov K; Borukhov S; Komissarova N; Goldfarb A
PMID: 8902816
ISSN: 0076-6879
CID: 17434
Coupling between transcription termination and RNA polymerase inchworming
Nudler E; Kashlev M; Nikiforov V; Goldfarb A
Advancement of RNA polymerase of E. coli occurs in alternating laps of monotonic and inchworm-like movement. Cycles of inchworming are encoded in DNA and involve straining and relaxation of the ternary complex accompanied by characteristic leaping of DNA and RNA footprints. We demonstrate that the oligo(T) tract that constitutes a normal part of transcription terminators acts as an inchworming signal so that the leap coincides with the termination event. Prevention of leaping with a roadblock of cleavage-defective EcoRI protein results in suppression of RNA chain release at a termination site. The results indicate that straining and relaxation of RNA polymerase are steps in the termination mechanism
PMID: 7736587
ISSN: 0092-8674
CID: 17435
Motion and enzymatic degradation of DNA in the atomic force microscope
Bezanilla M; Drake B; Nudler E; Kashlev M; Hansma PK; Hansma HG
The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length
PMCID:1225630
PMID: 7696484
ISSN: 0006-3495
CID: 17436
Discontinuous mechanism of transcription elongation
Nudler E; Goldfarb A; Kashlev M
During transcription elongation, three flexibly connected parts of RNA polymerase of Escherichia coli advance along the template so that the front-end domain is followed by the catalytic site which in turn is followed by the RNA product binding site. The advancing enzyme was found to maintain the same conformation throughout extended segments of the transcribed region. However, when the polymerase traveled across certain DNA sites that seemed to briefly anchor the front-end domain, cyclic shifting of the three parts, accompanied by buildup and relief of internal strain, was observed. Thus, elongation proceeded in alternating laps of monotonous and inchworm-like movement with the flexible RNA polymerase configuration being subject to direct sequence control
PMID: 8047884
ISSN: 0036-8075
CID: 17437
Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA
Kashlev M; Nudler E; Goldfarb A; White T; Kutter E
Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term 'memory' of recent pausing
PMID: 8402894
ISSN: 0092-8674
CID: 17438
Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli
Gragerov A; Nudler E; Komissarova N; Gaitanaris GA; Gottesman ME; Nikiforov V
Newly synthesized proteins aggregate extensively in Escherichia coli rpoH mutants, which are deficient in the heat shock proteins (hsp). Overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation. If expressed together, the four hsp are effective at physiological concentrations. Our data suggest that the GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins
PMCID:50334
PMID: 1359538
ISSN: 0027-8424
CID: 17439