Other studies identified an important contact of the linker region of TFIIB with the upstream single-stranded segment of the transcription bubble. This contact should be lost, and thus TFIIB-pol II interaction should be destabilized, once the upstream section of the transcription bubble reanneals during bubble collapse [ 5 ]. This question was recently addressed by directly assaying for loss of TFIIB from pol II early elongation complexes advanced in single nt increments [ 22 ].
This is well past the predicted initial collision point of TFIIB with the nascent RNA in the RNA exit channel, and crucially it is also 2 bp downstream of the point at which bubble collapse occurs on the promoter used [ 16 , 22 ]. The somewhat surprising retention of TFIIB beyond the point of bubble collapse suggests that additional important contacts govern the association of TFIIB in the transcription complex after initiation.
During promoter clearance, the initiation-specific interactions of pol II with the general transcript factors other than TFIIB should also be lost. TFIIF is unique among the general transcript initiation factors in that it can also associate with pol II throughout transcription and thereby stimulate the rate of transcript elongation [ 48 — 51 ].
TFIIF is transiently lost from the transcription complex after initiation [ 42 ]. The direct comparison of transcription with PICs that retained or lacked TFIIF showed no difference in the ability of those complexes to pass through the bubble collapse transition, indicating that TFIIF does not have a major influence on this phase of transcription [ 22 ]. The apparent discrepancy with the more recent work may be based on the different choice of template types: bubble templates were used for the Yan et al.
If bubble collapse marks the end of promoter clearance, the properties of the transcription complex should not change significantly downstream of collapse. At this point the complex should have acquired all of the stabilizing effects from the transcript-exit channel interactions.
RNA emerges from pol II at a transcript length of about 17 nt [ 54 , 55 ]. However, the properties of the complex continue to change during elongation, through the channel-filling process and ultimately well downstream of that point. For example, transcript slippage was evident in pol II complexes with mer and mer RNAs when the polymerase was forced to pause over a repetitive template segment [ 34 ]. Slippage was completely absent after pausing over the same repetitive element only when poi II had synthesized RNAs 23 nt or longer [ 34 ].
This is in contrast to mature pol II elongation complexes which do not backtrack when paused except at a very small subset of sequences that cause arrest [ 58 ]. It is not known why pol II complexes with 17—32 nt transcripts backtrack, nor is it understood why complexes with 12—13 bases upstream of the active site after backtracking are especially stable.
It is possible that secondary structure in the emerging RNA is important in blocking reverse threading of the transcript, thereby preventing backtracking once the RNA is of sufficient length. However, in the examples studied, no strong secondary structures could be predicted in the newly emerged transcripts for the complexes that no longer backtracked [ 57 ].
More importantly, hybridizing short DNA oligonucleotides to the transcripts at positions well upstream of the point of emergence of the RNA from the polymerase strongly increased backtracking and arrest [ 55 ], inconsistent with a model in which immediate self-association of the transcript is important to stabilize the transcription complex against backtracking. In the experiments just cited, the transcription complexes were detergent-rinsed and thus lacked any additional factors such as the capping machinery which could have bound to the emerging transcript and inhibited reverse threading of the RNA.
Thus, as judged by these properties, promoter clearance is complete for pol II by about 30 bases downstream of transcription start. While the clearance pathway just described has been reasonably well established for mammalian pol II at a TATA box promoter, it is important to acknowledge the limitations of this analysis.
Only a small minority of pol II promoters in mammalian cells contain TATA boxes and many mammalian promoters do not contain any known promoter sequence motifs recently reviewed in [ 37 , 59 , 60 ]. It is not yet known whether the canonical set of general initiation factors identified for TATA box promoters are either necessary or sufficient for initiation at non-TATA promoters see in particular [ 61 ].
In the absence of a TATA element to anchor the upstream end of the initial transcription bubble, it is not clear how the extension and ultimate collapse of the bubble would contribute to the clearance process.
The propagation of the initial transcription bubble in budding yeast is especially perplexing. Recent studies indicate that most S. In addition, the initial transcription bubble in yeast is apparently anchored at its upstream end to TATA, as in mammalian cells [ 15 ]. However, transcription in yeast can start over a wide range of permissible distances downstream from TATA: from 40 to bp [ 45 , 63 ], in contrast to the narrow 28—33 bp window in mammalian cells [ 37 ].
This makes it difficult to envision how expansion of the transcription bubble to a single maximum length would drive the clearance process in yeast, as hypothesized for mammalian pol II transcription [ 16 ]. Thus, it is not necessary to have an exceptionally long bubble within the open complex for yeast pol II to initiate transcription.
Work from Ponticelli and colleagues suggests that yeast pol II itself selects the transcript start site as it scans downstream from the initial, more TATA-proximal bubble position see [ 64 ] and [ 65 ] for more detailed discussion. The clearance pathway in Fig. During the clearance process in the nucleus, pol II transcription complexes will be modified and supplemented by the addition of many factors necessary to support effective elongation.
The acquisition of these modifications and factors, as well as the loading of additional factors involved in RNA processing, are reviewed in several of the accompanying papers in this issue. One point in particular is worth noting. Regulatory factors, often in conjunction with the Mediator coactivator complex, occupy the DNA upstream of the promoter of active genes see [ 66 ] for a recent review.
Association of pol II with Mediator and other factors provides additional stabilizing interactions for the transcription complex just after initiation, which could act to suppress abortive initiation. This stands in contrast to the case in prokaryotes where, as noted above, abortive production of short RNAs during the initial stages of transcription is evident both in vitro and in vivo. Interestingly, Mediator subunits associate not only with the promoter region but also with the bodies of actively transcribed genes, as determined by ChIP assays [ 67 ]; see also [ 68 ].
Based on these observations and the fact that Mediator can bind in a specific way to pol II [ 69 ], it is tempting to speculate that the transcription complex remains in contact with promoter-bound Mediator throughout the transcription process. In this model, pol Il presumably transitions into a stable elongation complex through a clearance path similar to that described above, but the polymerase also maintains continued association with the promoter through interactions with Mediator and other factors.
A mechanism for retaining some promoter contacts for pol II throughout the transcription cycle would allow polymerase to rapidly and effectively reinitiate transcription after termination. This is consistent with reports on the retention of subsets of the initiation factors at the promoter after initiation in yeast [ 71 ] and the connection of termination and initiation through looping interactions mediated in part by TFIIB [ 72 , 73 ].
Work from our laboratory discussed in this review was supported by grant GM from the National Institutes of Health. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.
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See other articles in PMC that cite the published article. Abstract Many changes must occur to the RNA polymerase II pol II transcription complex as it makes the transition from initiation into transcript elongation.
Introduction and terminology There is not a well-established terminology to describe the conversion of pol II from an initiation complex to a fully committed transcript elongation complex. The preinitiation complex and the initiation of transcription The preinitation complex PIC is the molecular assembly through which pol II can locate and utilize a promoter. Open in a separate window. Important stages in the progress of pol II to promoter clearance The downstream progress of the pol II transcription bubble from the initial open complex to the completely cleared elongation complex is shown schematically.
However, the interactions of damage is induced when cells are exposed to sublethal doses of RNA polymerase and Ada with these promoters differ. The product of the ada gene, Ada, is a methyl- methylated Ada meAda , and RNA polymerase at the transferase able to transfer methyl groups from damaged DNA alkA promoter and contrast these interactions with to two of its own cysteine residues 6, 7.
Methylation of Cys those characterized previously for the ada and aidB converts Ada into a transcriptional activator. The Cys promoters. In the presence of promoters, alkA and aidB, and activate their transcription either of these two activators, RNA polymerase protects 8 — RNA polymerase holoenzyme containing the a RNA polymerase a subunit results in the loss of transcriptional subunit mutation RA is severely impaired in Ada- activation by meAda at the ada promoter Binding of the purified wild type a ada and aidB in the absence of Ada protein RNA polym- subunit to alkA was not detected, but a complex of pro- erase holoenzyme binds the two promoter regions between moter DNA, Ada or meAda, and a was observed in gel and via its a subunit; this area overlaps the meAda binding shift assays.
This RNA polymerasezpromoter binary complex shows only basal levels of transcription and is modified by me Ada into a ternary complex competent in transcription initi- In bacteria, transcriptional activation plays a pivotal role in ation at induced levels The single amino acid substitution adaptation processes. This results in adaptation of rrnB P1 promoter 13 as well as the to region of the gene expression to the environmental requirements.
Transcrip- ada and aidB promoters According observations strongly suggest that the mechanism of Ada ac- to this definition, Class I transcriptional activators work by tion on the alkA promoter differs from that of the other adapt- contacting the a subunit of RNA polymerase, whereas Class II ive response genes.
First, methylation of Ada is not necessary activators interact with the s factor. Both Ada and meAda proteins are able to activate the and sequences, whereas a is able to bind a third transcription of alkA in vitro 9 , although higher concentra- element of the bacterial promoter identified at the rrnB P1 tions of Ada are required 15, In contrast, the ada and aidB promoters are only activated by meAda protein 6, 8 , and high concentrations of Ada protein inhibit activation by meAda The costs At the alkA promoter, meAda protects residues from to of publication of this article were defrayed in part by the payment of 16, 18 , overlapping the region.
In both ada and aidB, page charges. Section solely to indicate this me Ada binds farther upstream, protecting sequences from fact. North, Worcester, MA Volkert banyan. Promoter and upstream regions of the aidB, ada, and alkA genes. The double underlines indicate the proposed and regions of each promoter, as described in Landini and Volkert 10 aidB , Sakumi and Sekiguchi 19 ada , and Akimaru et al.
The single underline indicates the RNA polymerase binding sites in the absence of the Ada protein, as identified by DNase I protection assays The dashed overline indicates the meAda binding sites 10, 18, A plus or minus sign indicates the ability or inability of a or meAda to bind.
See text for further details. Third, Ada mutants capable of activating alkA but not ada, and transcripts were confirmed by DNA sequencing ladders run on the same vice versa, have been isolated 15, Methylation of Ada was performed as in Ref.
Transcription reactions were started by the addition of 0. Samples were precipitated with 2 volumes of ethanol, resuspended in gel loading DNA binding. Both the unmethylated and tion sites to obtain plasmid pMV To obtain pMV, pMV was the methylated forms of Ada Ada and meAda stimulated in cut with AccI, and the vector fragment was re-ligated to delete bp vitro transcription, although the highest levels of transcription from the polylinker of pSL We and others have bp fragment from pMV that contained the alkA promoter previously found that the carboxyl-terminal domain of the RNA region from to plus an additional 61 bp of upstream vector DNA was labeled at the AccI site with [aP]dATP by an end-filling polymerase a subunit plays a role at other Ada-activated pro- reaction with Klenow enzyme.
However, the binding site for meAda in the alkA EcoRI bp fragment from pMV that contained the same bp promoter to , partially overlapping the region fragment as pMV plus an additional 24 bp of downstream vector differs from that at the aidB and ada promoters that have DNA was labeled at the HindIII site.
The region downstream of me Ada binding sites extending from to and to contained all the necessary elements for transcription and regulation of , respectively. This result combined with the results from alkA Ref. Deletion of the region upstream of resulted in the loss of a second promoter that reads in the opposite the genetic and biochemical studies of others 11, 17, 18, 20 direction This promoter is not regulated by the Ada protein and suggests that the mechanisms at these promoters may differ.
DNase I protection and gel retardation experiments were performed in We used an RNA polymerase containing the a RA muta- 20 ml final volume of binding buffer This mutant a subunit was previously shown to be defec- In Vitro Transcription—Single-round in vitro transcription experi- tive in DNA binding and, when assembled into the RNA po- ments were performed using the linear DNA templates as follows.
Purified a RA was also shown to was used. This fragment contained the same bp alkA promoter be defective in the binding of the upstream regions of the fragment used in DNase I protection experiments of the template Ada-dependent aidB and ada promoters The a RA- strand. The RNA transcript obtained from this fragment was 48 nucle- otides in length. As a control, we used the lacUV5 promoter, a bp RNA polymerase was reconstituted in vitro and used in tran- fragment from pYN 9 , to produce an RNA transcript of 65 nucle- scription of alkA in the presence or absence of Ada and meAda otides.
In the experimental conditions, a second RNA transcript approx- Fig. The activator-independent, basal level of imately 10 nucleotides shorter was also produced, possibly from a transcription of alkA was reduced substantially by the RA cryptic promoter on the same DNA fragment.
The sizes of the RNA mutation lane 6 , and no activation of trancription was de- tected by either Ada or meAda lanes 7 and 8. Landini and M. Volkert, unpublished observations. Gel retardation experiments with Ada or meAda. Genes Dev. A code for transcription initiation in mammalian genomes. Development of a model for the prediction of transcription start site usage in mammals.
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