18 research outputs found
Transcriptional regulation in eukaryotic ribosomal protein genes
AbstractUnderstanding ribosomal protein gene regulation provides a good avenue for understanding gene regulatory networks. Even after 5 decades of research on ribosomal protein gene regulation, little is known about how higher eukaryotic ribosomal protein genes are coordinately regulated at the transcriptional level. However, a few recent papers shed some light on this complicated problem
Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast
<p>Abstract</p> <p>Background</p> <p>More than two thirds of the highly expressed ribosomal protein (RP) genes in <it>Saccharomyces cerevisiae </it>contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes.</p> <p>Results</p> <p>We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function.</p> <p>Conclusion</p> <p>Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.</p
Scoring functions for transcription factor binding site prediction
BACKGROUND: Transcription factor binding site (TFBS) prediction is a difficult problem, which requires a good scoring function to discriminate between real binding sites and background noise. Many scoring functions have been proposed in the literature, but it is difficult to assess their relative performance, because they are implemented in different software tools using different search methods and different TFBS representations. RESULTS: Here we compare how several scoring functions perform on both real and semi-simulated data sets in a common test environment. We have also developed two new scoring functions and included them in the comparison. The data sets are from the yeast (S. cerevisiae) genome. Our new scoring function LLBG (least likely under the background model) performs best in this study. It achieves the best average rank for the correct motifs. Scoring functions based on positional bias performed quite poorly in this study. CONCLUSION: LLBG may provide an interesting alternative to current scoring functions for TFBS prediction
Predicting Gene Expression from Sequence: A Reexamination
Although much of the information regarding genes' expressions is encoded in the genome, deciphering such information has been very challenging. We reexamined Beer and Tavazoie's (BT) approach to predict mRNA expression patterns of 2,587 genes in Saccharomyces cerevisiae from the information in their respective promoter sequences. Instead of fitting complex Bayesian network models, we trained naĆÆve Bayes classifiers using only the sequence-motif matching scores provided by BT. Our simple models correctly predict expression patterns for 79% of the genes, based on the same criterion and the same cross-validation (CV) procedure as BT, which compares favorably to the 73% accuracy of BT. The fact that our approach did not use position and orientation information of the predicted binding sites but achieved a higher prediction accuracy, motivated us to investigate a few biological predictions made by BT. We found that some of their predictions, especially those related to motif orientations and positions, are at best circumstantial. For example, the combinatorial rules suggested by BT for the PAC and RRPE motifs are not unique to the cluster of genes from which the predictive model was inferred, and there are simpler rules that are statistically more significant than BT's ones. We also show that CV procedure used by BT to estimate their method's prediction accuracy is inappropriate and may have overestimated the prediction accuracy by about 10%
The 19S proteasome subcomplex promotes the targeting of NuA4 HAT to the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional initiation in vivo
Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo
Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production
Thesis (MSc)--Stellenbosch University, 2019.ENGLISH ABSTRACT: The yeast, Saccharomyces cerevisiae, possesses multiple characteristics that classify it as an
excellent host organism for the recombinant production of industrial and medical proteins.
This includes its ability to process and secrete heterologous eukaryotic proteins at far higher
levels than native proteins, which significantly simplifies downstream protein purification.
However, it has had limited success in industry due to limitations in the yeastās recombinant
protein production capability.
Gene expression in S. cerevisiae is tightly regulated and metabolic engineering is therefore
required to direct cellular resources and metabolic energy towards heterologous gene
expression. Promoters are genetic elements that control gene expression and specific DNA
sequences within promoters act as binding sites for transcriptional activators. Transcriptional
activators upregulate gene expression, but they are also subject to regulation that could limit
the level of heterologous gene expression. Increasing the cellular concentration of such
activators could further upregulate gene expression and consequently, increase recombinant
protein production.
The aim of this study was to develop a tool for the overproduction of recombinant proteins in
S. cerevisiae and increase its competitiveness as a microbial cell factory in industry.
Therefore, the effect of overexpressing two promoter-specific transcriptional activators
(RAP1 and GCR1) on the strength of three strong constitutive promoters (ENO1P, ATEF1P
and STEF1P) in the S. cerevisiae Y294 laboratory strain, was evaluated. Reporter genes
coding for raw starch-degrading enzymes were used as they have a large industrial
significance in the food, fermentation, textile, paper, detergent and pharmaceutical industries.
The abovementioned transcriptional manipulations were evaluated in terms of the
extracellular enzyme activity and protein production under aerobic and fermentative
conditions. The best candidates were further tested in raw corn starch fermentations in terms
of ethanol production and carbon conversion.
While GCR1 overexpression had no effect, RAP1 overexpression resulted in a three-fold
increase in the extracellular protein concentration. Under aerobic conditions, the volumetric
activity of the recombinant AteA Ī±-amylase was increased by 81%, 28% and 46%, when the
gene was expressed under the transcriptional control of the ENO1, ATEF1 and STEF1
promoters, respectively. In addition, improvements of 99%, 29% and 69% were observed
under fermentative conditions for the three respective promoters. RAP1-overexpressing
strains were subsequently used for the construction of an amylolytic co-culture (S. cerevisiae
Y294[ENO1_TemA_RAP1] + Y294[ENO1_TemG_RAP1]) that exhibited an improved
fermentation profile during the bioconversion of 200 g/l raw corn starch. The ethanol yield
produced after 48 hours was comparable to that obtained from a commercial enzyme
preparation (STARGENTM 002) and 54% higher than the benchmark co-culture
(S. cerevisiae Y294[ENO1_TemA] + Y294[ENO1_TemG]). More encouraging, was that the
ethanol yield from the S. cerevisiae Y294[TemA_TemG_RAP1] strain (co-expressing the
TemA and TemG genes) was 111% higher with RAP1 overexpression and 201% higher than
that of the S. cerevisiae Y294[AmyA_GlaA] reference strain after 48 hours. Furthermore,
RAP1 overexpression resulted in a fermentation profile in the S. cerevisiae
Y294[TemA_TemG_RAP1] laboratory strain that was comparable to that of a recombinant
S. cerevisiae industrial strain (S. cerevisiae Ethanol Red[TemA_TemG]).
This study has confirmed the potential of RAP1 overexpression as a tool to increase
recombinant protein production in S. cerevisiae, thus increasing the economic viability of
S. cerevisiae as a microbial cell factory.AFRIKAANSE OPSOMMING: Die gis, Saccharomyces cerevisiae, bevat vele eienskappe wat dit 'n uitstekende mikrobiese
gasheer vir die produksie van rekombinante industriĆ«le en medies-toepaslike proteĆÆene maak.
Dit sluit in die vermoĆ« om heteroloĆ« eukariotiese proteĆÆene te prosesseer en teen baie hoĆ«r
vlakke as eie proteĆÆene uit te skei, wat stroom-af suiwering van proteĆÆene beduidend
vereenvoudig. Dit het egter tot dusver beperkte sukses in die industrie gehad weens
beperkinge in die gis se rekombinante proteĆÆenproduksievermoĆ«.
Geenuitdrukking in S. cerevisiae word streng gereguleer en metaboliese ingenieurswese word
dus vereis om sellulĆŖre hulpbronne en metaboliese energie na heteroloĆ« geenuitdrukking te
herlei. Promotors is genetiese elemente wat geenuitdrukking beheer en spesifieke DNA-
volgordes in promoters funksioneer as bindingsetels vir transkripsionele aktiveerders.
Transkripsionele aktiveerders verhoog geenuitdrukking, maar hulle is self ook aan regulering
onderworpe wat die vlak van heteroloƫ geenuitdrukking kan beperk. 'n Verhoging in die
sellulĆŖre konsentrasie van sulke aktiveerders kan moontlik geenuitdrukking bevorder en dus
die vlakke van rekombinante proteĆÆenproduksie verhoog.
Die doel van hierdie studie was om gereedskap vir die oorproduksie van rekombinante
proteĆÆene in S. cerevisiae te ontwikkel en sodoende hierdie gis se mededingendheid as 'n
mikrobiese selfabriek in die bedryf te verhoog. Die effek van die ooruitdrukking van twee
promotor-spesifieke transkripsionele aktiveerders (RAP1 en GCR1) op die sterkte van drie
sterk konstitutiewe promotors (ENO1P, ATEF1P en STEF1P) is in die S. cerevisiae Y294
laboratoriumras geƫvalueer. Verklikkergene wat vir rou stysel-hidroliserende ensieme kodeer
is hiervoor gebruik, aangesien hulle groot industriƫle waarde in die voedsel-, fermentasie-,
tekstiel-, papier-, skoonmaakmiddel- en farmaseutiese bedryf het. Bogenoemde
transkripsionele manipulasies is in terme van die ekstrasellulĆŖre ensiemaktiwiteit en
proteĆÆenproduksie onder aĆ«robiese en fermentatiewe toestande geĆ«valueer. Die beste
kandidate is verder in rou mieliestysel fermentasies in terme van etanolproduksie en koolstof-
omskakeling getoets.
Terwyl GCR1-ooruitdrukking geen effek gehad het nie, het RAP1-ooruitdrukking die
rekombinante proteĆÆenkonsentrasie tot drievoudig verhoog. Onder aĆ«robiese toestande is die
volumetriese aktiwiteit van die rekombinante AteA Ī±-amilase met 81%, 28% en 46%
verhoog toe die geen onderskeidelik onder die transkripsionele beheer van die ENO1, ATEF1
en STEF1 promotors uitgedruk is. Verder is verbeterings van 99%, 29% en 69% vir die
onderskeie promotors onder fermentatiewe toestande waargeneem. Ooruitdrukking van RAP1
is gevolglik vir die konstruksie van 'n amilolitiese kombinasiekultuur (S. cerevisiae Y294
[ENO1_TemA_RAP1] + Y294[ENO1_TemG_RAP1]) gebruik wat gedurende die bio-
omskakeling van 200 g/l rou mieliestysel 'n verbeterde fermentasieprofiel getoon het. Die
etanolopbrengs was vergelykbaar met 'n kommersiƫle ensiempreparaat (STARGENTM 002)
en 54% hoƫr as die maatstaf kombinasiekultuur (S. cerevisiae Y294[ENO1_TemA] +
Y294[ENO1_TemG]). Meer bemoedigend, was die feit dat die etanolopbrengs van die
S. cerevisiae Y294[TemA-TemG_RAP1] ras (wat die TemA and TemG gene gesamentlik
uitdruk) na 48 ure 111% hoƫr met RAP1 ooruitdrukking en 201% hoƫr as die S. cerevisiae
Y294[AmyA-GlaA] verwysingsras was. Verder het die ooruitdrukking van RAP1 ān
fermentasieprofiel in die S. cerevisiae Y294[TemA_TemG_RAP1] laboratoriumras geskep
wat vergelykbaar met 'n industriƫle ras (S. cerevisiae Ethanol Red[TemA_TemG]) was.
Hierdie studie het die potensiaal van RAP1-ooruitdrukking as 'n hulpmiddel vir verhoogde
rekombinante proteĆÆenproduksie in S. cerevisiae bevestig, wat sodoende die ekonomiese
lewensvatbaarheid van S. cerevisiae as 'n mikrobiese selfabriek kan verhoog.Master