scholarly journals Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.

1984 ◽  
Vol 4 (4) ◽  
pp. 657-665 ◽  
Author(s):  
K J Shaw ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Deletion Mutants ◽  
Flanking Sequence ◽  
Base Pairs ◽  
In Vivo Expression ◽  
Flanking Sequences

Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation.

Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene

1984 ◽  
Vol 4 (4) ◽  
pp. 657-665
Author(s):  
K J Shaw ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Deletion Mutants ◽  
Flanking Sequence ◽  
Base Pairs ◽  
In Vivo Expression ◽  
Flanking Sequences

Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation.

scholarly journals Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751 ◽  
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

2001 ◽  
Vol 21 (19) ◽  
pp. 6429-6439 ◽  
Author(s):  
Michael P. Martin ◽  
Valerie L. Gerlach ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Tata Box ◽  
Initiation Factor ◽  
Base Pairs ◽  
Polymerase Iii ◽  
U6 Rna

ABSTRACT The Saccharomyces cerevisiae U6 RNA gene,SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription ofSNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position −30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele ofSNR6 with decreased spacing between the A and B blocks,snr6-Δ42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Δ42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Δ42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription ofSNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

scholarly journals Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839 ◽  
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals The Yeast hnRNP-Like Proteins Yra1p and Yra2p Participate in mRNA Export through Interaction with Mex67p

2001 ◽  
Vol 21 (13) ◽  
pp. 4219-4232 ◽  
Author(s):  
Daniel Zenklusen ◽  
Patrizia Vinciguerra ◽  
Yvan Strahm ◽  
Françoise Stutz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Protein ◽  
Rna Binding ◽  
Mrna Export ◽  
Deletion Mutants ◽  
Variable Regions ◽  
Evolutionarily Conserved ◽  
Rna Export

ABSTRACT Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessentialSaccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)+ RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

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