scholarly journals The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion

2012 ◽  
Vol 20 (2) ◽  
pp. 174-181 ◽  
Author(s):  
Feng Wang ◽  
Sy Redding ◽  
Ilya J Finkelstein ◽  
Jason Gorman ◽  
David R Reichman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Three Dimensional ◽  
Dimensional Diffusion ◽  
Search Mechanism

scholarly journals One-dimensional sliding assists σ70-dependent promoter binding by Escherichia coli RNA polymerase

2018 ◽  
Author(s):  
Iddo Heller ◽  
Margherita Marchetti ◽  
Abhishek Mazumder ◽  
Anirban Chakraborty ◽  
Agata M. Malinowska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Three Dimensional ◽  
Optical Traps ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Single Dna Molecules ◽  
Search Mechanism ◽  
Dna Base

ABSTRACTThe search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial. Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP σ70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP σ70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the “diffusion facilitation threshold” – the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding – of 0.2 μM RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 μM, concentrations of RNAP in cells.Significance statementThe flow of genetic information from DNA to RNA is of central importance to living systems, and it can only start after an RNA polymerase (RNAP) has found a promoter site. But how does this enzyme find promoter sites on DNA in the first place? In recent years, debate on this topic has favored a promoter search mechanism that is dominated by three-dimensional diffusion of RNAP, rather than by one-dimensional sliding of RNAP on DNA. Here, we designed an improved single-molecule assay that unambiguously revealed extensive one-dimensional sliding of RNAP on DNA. Our results imply that, at the RNAP concentrations in living cells, the promoter-search process is facilitated by one-dimensional sliding on DNA.

scholarly journals Direct Observation of One-Dimensional Diffusion and Transcription by Escherichia coli RNA Polymerase

1999 ◽  
Vol 77 (4) ◽  
pp. 2284-2294 ◽  
Author(s):  
Martin Guthold ◽  
Xingshu Zhu ◽  
Claudio Rivetti ◽  
Guoliang Yang ◽  
Neil H. Thomson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Direct Observation ◽  
One Dimensional ◽  
Dimensional Diffusion

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 166-167 ◽  
Author(s):  
G. Stöffler ◽  
R.W. Bald ◽  
J. Dieckhoff ◽  
H. Eckhard ◽  
R. Lührmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Spatial Arrangement ◽  
Small Subunit ◽  
Antibody Binding ◽  
Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

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