Transcription in prokaryotes Elongation and termination After initiation the σ factor leaves the scene. Core polymerase is conducting the elongation of the chain. The core polymerase contains main nucleotide synthesizing machinery: β - involved in phosphodiester bond formation β and β involved in DNA binding α- has several actions, including assembly of polymerase. Role of subunits were first investigated in 1970 by Walter Zillig 1
Role of core subunits Approach: separating core into polypeptides and combining them back to reconstitute the functions. Separation: electrophoresis on cellulose acetate with the presence of urea. Urea is denaturing agent that can separate individual polypeptides from the complex. Advantage: It is a mild agent, easy to remove. Separation and reconstitution allowed to figure out which subunit determined resistance or sensitivity to rifampicin. α+β+ σ from rif sensitive bacterium + β from rif resistant = rif resistant polymerase 2
β subunit is involved in PDB formation Approach: used affinity labeling experiment Idea: Label an enzyme with a derivative of a normal substrate that can be cross-linked to protein. The affinity reagent can seek out and label the active site of the enzyme. The enzyme can be dissociated, and one can see the subunit that the tag was attached to. 14 different reagents were used all ATP and GTP analogs. Outcome: β subunit is the only core subunit labeled by the affinity reagents, suggesting that it is responsible for nucleotide binding and PDB formation. Conclusion: β subunit is involved in PDB formation One of different reagents that were used. The ATP analog. Steps: 1. Add analog to RNA polymerase. It covalently binds to amino groups at active site 2. Add radioactive UTP, it forms PDB with the enzyme-bound reagent. Fig. 6.32 3. Reaction occurs at the active site which becomes labeled. Outcome: β subunit is the only core subunit labeled by the affinity reagents, suggesting that it is responsible for nucleotide binding and PDB formation. 3
Conclusion: Subunit β binds nucleotides at the active site of the RNA polymerase where PDBs are formed The σ factor is also near the nucleotide-binding site, at least during initiation. β and β and DNA binding Studies of E. Nudler, 1996 Cross-linking labeled DNA to polymerase They have concluded: β and β are involved in DNA binding Two binding sites are present An upstream, weak site where melting occurs, with electrostatic forces predominating Downstream strong site where hydrophobic forces bind DNA and protein together 4
Role of α subunit in polymerase assembly The α Subunit recognizes UP elements It also interacts with activator proteins that bind outside of promoter The N terminal domain plays an important role in transcription by helping to organize the polymerase. How? Order of assembly: α α2 α2 β α2ββ - possibly α plays central role? Role confirmed by studies involving mutant E. coli strains Role of α subunit was confirmed by studies involving mutant E. coli strains Mutants with deletions at C-terminus of α were studied Mutant proteins were terminated at aa 150,176, 235, 256 and 296. Wt protein terminates at 329. Outcome: up to 94aa could be removed from C-terminus w/o loss of function Conclusion: Assembly function is in the N-terminus. The points of contact between the α -NTD and the β and β subunits lie on the opposite side of the α -NTD dimmer from the dimmer s C-terminal domains. 5
Elongation The elongation complex Experiments with RNA-DNA hybrids The RNA-DNA hybrids within the elongation complexes extend from position 1 to 9,-8 with respect to the 3 end of the emerging RNA. The processivity of transcription depends upon the DNA- RNA hybrid at least 9bp long. Fig. 6.49 6
From Genes VII web resource www.oup.com/genesvii Termination 7
RNA pol moves along the DNA synthesizing RNA until it meets a terminator (t) sequence. At this point the polymerase: Stops adding nucleotides Releases the completed product Dissociates from the template. NB we do not know in which order the last two happen. Termination requires that all hydrogen bonds holding DNA-RNA hybrid together must be broken, after which the DNA duplex reforms. Termination point Is difficult to define the term. point at the RNA that has been synthesized in the living cell. Because it is always possible that the 3 end of the molecule has been generated by cleavage of the primary transcript, and therefore does not represent the actual site at which pol was terminated. The best identification of the terminator sites is provided by in vitro systems. But: because ability of the enzyme to terminate is strongly influenced by parameters such as ionic strength, the term point in vitro does prove that the same point is a natural terminator. 8
Anti-termination will cause enzyme to continue past the t sequence the read-through. The hairpin structure From GenesVII web resource www.oup.com/genesvii 9
Termination is viewed not as a simple event to stop transcription, but as an opportunity to control gene expression. Termination, as initiation, is a subject to specific control. Parallels between initiation and termination: Both require breaking of hydrogen bonds Both requires additional proteins to interact with core enzyme. Differences: Initiation relies solely on interaction between DNA and pol Termination involves recognition of signals in the transcript by RNA pol and ancillary enzymes Sequences of prokaryotic terminators show no similarities beyond the point at which the last base is added to the RNA. The responsibility for termination is with the sequences already transcribed by RNA polymerase. It relies on scrutiny of the template or product that polymerase is currently producing. 10
Terminators: Core enzyme can terminate in vitro at certain sites in absence of any other factors. These sites are called intrinsic terminators. Rho-dependent terminators are defined by the need of addition factor (ρ) in vitro. Intrinsic terminators include palindromic regions The stem-loop structure with a G-C rich region. a run of U-nucleotides. 11
What is the effect of hairpin? Probably the hairpins cause the polymerase to slow or perhaps to pause the synthesis. Pausing creates the opportunity for termination. The length of the pause varies, but at a typical terminator it lasts ~60 seconds. Role of the U-run A U-string is necessary for polymerase to dissociate from the template when it pauses at the hairpin. The ru-da RNA-DNA hybrid is unusually weak bp structure. Termination usually takes place at any of several positions toward or at the end of U-run, as though the enzyme stutters during termination. Importance of the U-run was confirmed by making deletions that shorten the stretch although the polymerase paused, it could not terminate. 12
The model Fig. 6.51 The Rho factor how does it work The Rho factor: Functions in E. coli solely at the termination stage. 46kD protein, acts as hexamer. Function: ancillary factor of RNA polymerase. Max activity displayed in vitro when present at conc. ~10% of RNA polymerase. E. coli has relatively few Rho factors. The sequences required for Rho are upstream of terminator, are 50-90 bp long. Common feature the RNA is rich in C and poor in G residues. EG: C-41%, g-14%. Rule: efficiency of Rho terminator depends upon increases in length of the C-rich/G-poor region. 13
The Rho factor how does it work From GenesVII web resource www.oup.com/genesvii The Rho factor how does it work Rho factor: Has ATPase activity Binds to RNA An individual Rho factor acts processively on the RNA strand The hot pursuit model 14
The Rho factor how does it work Fig. 6.56 The Rho factor how does it work From GenesVII web resource www.oup.com /genesvii 15
The action of Rho may create a link between transcription and translation Rho first must have access to a binding sequence of RNA Must be able to move along the RNA Either of both of these conditions may be prevented if ribosomes are translation an RNA. Thus the ability of Rho factor to reach a terminator depends upon what is happening in translation The model to explain this puzzling phenomenon: In some cases, nonsense mutation in one gene of transcription unit prevents the expression of subsequent genes in the unit. This effect is called polarity. A common cause is the absence of mrna corresponding to the distant parts of the unit. Example: Suppose there are Rho-dependent terminators within the transcription unit, that is before the terminator that is usually used. Normally these early terminators are not used, because the ribosomes prevent Rho from reaching the RNA polymerase. But nonsense mutation releases the ribosomes, so that rho is free to attach and move along the RNA, enabling is to react with RNA polymerase. Result: enzyme is released, distal parts are not transcribed.???why there should be internal terminators??????why there should be internal terminators??? Perhaps they are simply sequences that by coincidence mimic the usual rho-dependent terminator. 16
From GenesVII web resource www.oup.com /genesvii Phenomenon of antitermination: Antitermination proteins allow RNA polymerase to transcribe through certain termination sites. Is used as a control mechanism in phage regulatory circuits and bacterial operons. 17
References: R. Weaver. Molecular Biology B. Lewin. Genes VII, 2000, web resource: www.oup.com/genesvii Reading: Elongation. Pages 154-171. W/o extensive details of crystallography studies. Termination: Pages 171-178 W/o details of figure 6.52 18