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. 1998 Jan;18(1):276-89.
doi: 10.1128/MCB.18.1.276.

Poly(A)-driven and poly(A)-assisted termination: two different modes of poly(A)-dependent transcription termination

G Yeung  1 , L M Choi, L C Chao, N J Park, D Liu, A Jamil, H G Martinson
Affiliations

Poly(A)-driven and poly(A)-assisted termination: two different modes of poly(A)-dependent transcription termination

G Yeung et al. Mol Cell Biol. 1998 Jan.

Abstract

We mapped the elements that mediate termination of transcription downstream of the chicken betaH- and betaA-globin gene poly(A) sites. We found no unique element and no segment of 3'-flanking DNA to be significantly more effective than any other. When we replaced the native 3'-flanking DNA with bacterial DNA, it too supported transcription termination. Termination in the bacterial DNA depended on a functional poly(A) signal, which apparently compelled termination to occur in the downstream DNA with little regard for its sequence. We also studied premature termination by poorly processive polymerases close to the promoter. The rate of premature termination varied for different DNA sequences. However, the efficiencies of poly(A)-driven termination and promoter-proximal premature termination varied similarly on different DNAs, suggesting that poly(A)-driven termination functions by returning the transcription complex to a form which resembles a prior state of low processivity. The poly(A)-driven termination described here differs dramatically from the poly(A)-assisted termination previously described for the simian virus 40 (SV40) early transcription unit. In the SV40 early transcription unit, essentially no termination occurs downstream of the poly(A) site unless a special termination element is present. The difference between the betaH-globin and SV40 modes of termination is governed by sequences in the upstream DNA. For maximum efficiency, the betaH-globin poly(A) signal required the assistance of upstream enhancing sequences. Moreover, the SV40 early poly(A) signal also drove termination in betaH-globin style when it was placed in a betaH-globin sequence context. These studies were facilitated by a rapid, improved method of run-on transcription analysis, based on the use of a vector containing two G-free cassettes.

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Figures

FIG. 1
FIG. 1
Vectors for studying transcription termination and representative experiments to illustrate their use. (A) pORgf3·2, the principal transient-expression plasmid used in this work to study transcription termination. “3·2” signifies a 376-nucleotide cassette, followed by a 261-nucleotide cassette. Insertion derivatives of pORgf3·2 are named as follows for insert X in the upstream multiple cloning site and insert Y between the cassettes: pORgf3·2:X. For brevity, we often abbreviate this as pX3·2 or simply pX. (B) Comparison of terminating and nonterminating inserts by G-free cassette analysis. The uncorrected readthrough values shown are the postsequence/presequence (Post/Pre) ratios normalized for C content (175 and 112) in the long and short cassettes, respectively. For reasons we do not yet understand, there is some variation in the postsequence/presequence ratio for any given plasmid from one transfection to another. This source of experimental uncertainty can be substantially reduced by including in each set of transfections a standard reference plasmid against which all transfectants are normalized. The normalization given here was to pORgf3·2. (C) pHyb, a conventional plasmid for studying termination by run-on transcription and hybridization analysis. (D) Comparison of terminating and nonterminating inserts by hybridization analysis. The uncorrected readthrough is calculated for C contents of 107 and 203 for pre- and postsequences, respectively. Normalization was to pHyb itself.
FIG. 2
FIG. 2
Maps of the βH- and βA-globin gene transcription termination regions drawn to scale and calibrated in base pairs. The run-on transcription endpoints indicated are those of Pribyl and Martinson (50) for βH-globin and of Villeponteau et al. (63) for βA-globin. The βH-globin map is of the DNA insert in clone p<ABCDEF>. Relevant restriction sites are shown. All are native chicken sites, except for the flanking BamHI and XbaI sites and the internal XhoI site. The βA-globin map is of the DNA insert in p<WXYZ>. Restriction sites shown are native chicken sites, except for the flanking SmaI and XbaI sites. The lettered segments in subclones used in this work (e.g., in Fig. 5 through 7) correspond to the lettered segments shown below the corresponding map, although for some constructs (where described) we used segments of slightly differing lengths. The exact cloning strategies for all clones are described in Materials and Methods and Table 1.
FIG. 3
FIG. 3
(A) The T1-resistant background is of cellular origin. Cells were either transfected with pORgf3·2 (+) or left untransfected (−) and then processed for G-free analysis in the usual way. (B) G-free transcription is α-amanitin sensitive, but the cellular background is α-amanitin resistant. Cells were transfected with pAsv<Bsv>, and the subsequent nuclear preparation was divided in two. Each part was then subjected to run-on transcription in the presence (+) or absence (−) of α-amanitin at 0.12 μg/ml. The region of the gel exhibiting the 261-nucleotide cassette transcript is shown. pAsv<Bsv> does not contain a 376-bp cassette. (C) Survival of 750-nucleotide G-free transcripts in the standard G-free run-on procedure. Both the image of the gel lane and its associated scan are shown. The transfecting plasmid was pORgf7. (D) Effect of omitting GTP from the cold chase in the standard G-free run-on procedure. An aliquot of nuclei from the same nuclear preparation as that used for panel B was subjected to run-on transcription in parallel with panel B, except that GTP was omitted from the cold chase. Braces indicate a cluster of background bands referred to in the text.
FIG. 4
FIG. 4
Comparison of the G-free and hybridization methods of transcription termination analysis. Readthrough values were normalized to that of p. Error bars show standard deviations, and the number of independent measurements taken for each point is given below.
FIG. 5
FIG. 5
Dissection of the βH- and βA-globin transcription termination regions. The percent readthrough was determined by the G-free run-on procedure, and values were normalized to that of pD<>. (A) Deletion analysis of the βH-globin region. (B) Modular rearrangements of the βH-globin region. (C) Deletion analysis of βA-globin. Error bars show standard deviations.
FIG. 6
FIG. 6
Transcription termination by RNA polymerase II in prokaryotic DNA. (A) Standard constructs with the βH-globin poly(A) site. (B) Split constructs with the βH-globin poly(A) site. (C) Split constructs with the SV40 poly(A) site. For panels A through C, normalization was to pD<>. (D) Various arrangements of G-free sequences. The plasmids in lanes 1 and 2 were pORgf3·2 itself (also called p<>) and its AB derivative, respectively. The plasmids in lanes 3 through 5 were derivatives of pORgf1·2. For this panel, each plasmid was first normalized to its homologous parent transfected in parallel and then this value was multiplied by the readthrough ratio (p<>/pD<>). Thus, the value for lane 2 was normalized to that of pORgf3·2 and the value for lane 1 was normalized to itself. The values for lanes 3 through 5 were normalized to that for pORgf1·2. The readthrough value for pD<> was assumed to reflect complete readthrough of both cassettes. Multiplying by the p<>/pD<> ratio allowed direct comparison to all of the other data in this report. Error bars show standard deviations.
FIG. 7
FIG. 7
Termination and enhanced termination promptly after the poly(A) site. (A) Termination downstream of the poly(A) site. (B) Termination enhancer in the βH-globin region. Normalization was to pD<>. Error bars show standard deviations.
FIG. 8
FIG. 8
Chicken βH-globin and SV40 early transcription units demonstrate two modes of transcription termination. Lanes 1 and 3 are identical to lane 5 of Fig. 7B and lane 6 of Fig. 6B, respectively. Normalization for lane 2 was to pAsv<Bsv>. All other lanes were normalized to pD<>. Error bars show standard deviations.
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