Download Determination of the Nucleotide Sequence Which Affects on the

Mol. Cells, Vol. 3, pp. 403-406
Determination of the Nucleotide Sequence Which Affects
on the
Transcription Initiation Site of Yeast rRNA Gene
Hyuk Ran Kwon, Jae Hyun Kim, Chan Hee Lee l , Hung Sun Koh and
Soo Young Choe*
Department of Biology and (Department of Microbiology, Chungbuk National University,
Chungbuk, Cheongju 360-763, Korea
.
(Received on October 8, 1993)
We have previously reported that the Sacchromyces cerevisiae ribosomal gene promoter consists
of at least two essential domains, an upstream domain located at the 5' boundary near position
-150 and a core domain around the transcription initiation site at + 1 (Choe et ai., 1992).
We also showed that the yeast ribosomal gene promoter has a critical requirement for binding
of protein or protein complex to core and upstream domains to be located at precise positions
on the face of the DNA helix (Rho et al., 1993). Here we show the promoter activity and
the position of transcription initiation site are highly dependent on the nucleotide sequences
neighboring the original + 1 site. The substitution of the original + 2 nucleotide (T) to C
. or G shifted the transcription initiation site to - 1, but the substitution to A did not. The
mutation of the sequences from + 13/ + 17 only affected on the promoter activity, not on
the detennination of tanscription initiation site. These results suggest that the transcription
initiation site of the ribosomal gene promoter requires a typical arrangement of nucleotides
around the + 1 position.
The 35S ribosomal RNA genes in yeast, Saccharom yces cerevisiae, are known to contain two DNA elements which affect on the transcription initiation by
RNA polymerase I. These are the 'gene promoter' ,
located at the 5' end of the 35S coding region, and
the 'enhancer' , an element at the 3' end of the 35S
coding region. The location of these two transcription
control elements is diagrammed in Figure lA. The
enhancer was originally described as a 180 bp EcoRIto-HindIII fragment close to 3' end of the 35S precursor by in vivo studies (Elion and Warner, 1984, 1986).
Recently we reported that the enhancer fragment reproducibly can stimulate ribosomal gene promoter activity under the proper in vitro conditions, and that the
enhancer functions only to assist stable complex formation (Schultz et al., 1993). In vivo (Musters et al.,
1989) and in vitro (Kulkens et aI., 1991 ; Choe et aI.,
1992; Rho et al., 1993) analysis of the gene promoter
shows that it consists of about 150 base pairs (bp)
and that it can be separated into core and upstream
domains which seem to be necessary for positioning
of transcription initiation factors on the correct face
of the DNA. In this study we show that the transcription initiation site can be changed by substitution of
the nucleotides neighboring the original + I site.
The plasmid pYrllA (Rho et al., 1993) was used
for the construction of all the promoter mutation plasmids in this study. The substitutions of the +2 nucleotide, the introducing of the BssHII or SmaI site into
Materials and Methods
the· diagram shows the single strand DNA used as DNA
probe for Sl assay. (C) Sequences of the ribosomal gene
promoter. The promoter region was subcloned and tagged
by inserting an Xhol linker (underlined) into the Taql site
25 bp downstream from the transcription start site.
Plasmid constructs
* To
whom correspondence should be addressed.
A.
B.
«-co
-
~~ ~JS
~
-=---
''''''''''''ItI( :~~~rJ+:1-x
Gene promoter
=:J---
~
) ,'+
" -
c.
- - - - - 5'
pGEM3·EX
SI ossay probc (Kpnl-Xhol single strand)
·200
I
CCCGGGGCACCfGTCACTTTGGAAAAAAAAAAGAAAAAAATATACGCfAA
s-J
-ISO
GATITTTGGAGAATAGCTTAAATTGAAGTITTTCf CGGCAAGAAATACG T
. 100
AGTTAAGGCCGAGCGACAcAGAGGGCAAAAGAAAATAAAAGTAAGATTTT
.S<)
AGTTTGTAATGGGAGGGGGGGTTTAGTCATGGAGTACAAGTGTGAGGAAA
+1
AGTAGTTGGGAGGTACTTCATGCGAAAGCAGTTGAAGACAAGTTCGTCAAGACCCfCGAG
""'"
Figure 1. (A) Diagram of the yeast ribosomal gene repeat.
(B) Diagram of the minigene for Sl assay. The arrow under
©
1993 The Korean Society of Molecular Biology
404
Yeast Ribosomal RNA Transcription
the promoter were made by oligonucleotide-directed
mutagenesis (Kunkel, 1985). The promoter region of
35S ribosomal DNA in pYrllA was subcloned into
M13mpl8 DNA and uracil containing single-stranded
recombinant DNA was prepared by using CJ236
(ung - ) cells. The oligonucleotides used for substitution
of +2 nucleotide and for making BssHII and Sma I
sites are 5'-CTGCTTTCGC(TCG)TGAAGTACC-3',
5'-CGAACTTGTCGCGCGCTGCTTTCGC-3', a nd
5'-CTTGTCTTCCCCGGGTTTGGGATGAAGTACC3', respectively (synthesized and purchased from the
Institute for Molecular Biology and Genetics, Seoul
National University). After confirming th at the recombinant M13 phages have the desired mutations, the
promoter region resubclol1ed into the pGEM3-Xho
plasmids.
In vitro transcription assays
Yeast extract prepa ration, in vitro tra nscription reactions, and S 1 nuclease protection assays were performed by the procedures of Rho et al. (1993) and Labhart and Reeder (1986). The Sl nuclease protection
probes for transcription were single-stranded DNAs
from Kpn I-XhoI fragments of the templ ate pl asm ids
(Fig. IB). After digestion of the plasm ids containing
the muta tion in promoter region, the DNA fragments
-were 5' end-labeled by P 32_[ y-ATP]' The labeled DNA
strands were separated in preparative 8% acrylamide
gel, a nd used for the hybridization with the RNA transcripts from the pl as mids. S 1 nuclease digestion o f
the hybridized materials should give 40 nucleotides
long fragments at the case of wild type plasmids.
Quantitation of the transcription was carried by measuring the intensity of the bands in X-ray film using
densitometer (LKB 2202 Ultroscan Laser Densito meter, Sweden).
Mol. Cells
Yeast rONA 3' mutants
+'
I
+10
+20
+3 0
+40
I
I
I
I
+10
+20
+30
+40
I
I
I
I
+10
+20
+30
+40
I
I
I
TT
TC
TG
TA
AGT AGTTGr.GAGGTACT TCA TGCGAAAGCAGT TGAAGACAAGT Tcgt caagaccctcqaq
C
Xhol
T - Bss H
C-B ss H
G-B ss H
A-B ss H
AGT AGT TGGGAGGTACTTCA TGCGAAAGCAGCGCCCGACAAGTT cgtcaagaccctc9a9
C
Bss H11
Xho l
G
A
LS +2 / + 15
AGTAGT TGGGAGGTACT TCAT£C£AAA£C£GQl!GAAGACAAGTT cgtcaagacc~
+1
I
+1
I
Smal
I
Xhol
Figure 2. DNA sequences of the mutated promoters. More
details are described in Results and Discussion. All of the
mutations were made by oligonucleotide-directed mutagenesis by the method of Kunkel (1985).
MI234
45 base (-5 ) 40 base (+ 1) -
5
678M
• ••
Figure 3. Transcription signal from the templates which contain one nucleotide change at +2 site. The single strand
probe DNAs were isolated after the plasm ids were digested
with Kpnl /X ho l restriction enzymes. Lane M, size marker
of 45 bp; lanes I and 5, transcription signal from wild type
promoter; lanes 2 and 6, transcription signal from the template which has T~C mutation at + 2 site; lanes 3 and 7,
same as above except T~G mutation ; lanes 4 and 8. same
as above except T~A mutation.
Results and Discussion
Transcription initiation site can be m oved by changing
the nucleotide at position +2
Previous in vivo and in vitro studies have shown
that yeast ribosomal genes produce a major transcript
initiating with an adenine residue (+ I in Fig. I C).
We reported that the 5' boundary of the promoter
was loca ted at betwee n - 158 a nd - 145. a nd th at
the promoter can be separa ted into core a nd upstrea m
domains and these two domains seem to be necessary
for positioning of tra nscription initiation factors on
the correct face of th e DNA (Choe et al., 1992; Rho
et al., 1993).
We have altered the nucleotide at + 2 thymidine
(T) to C, G , or A (Fig. 2). The purpose of this experi ment is whether the transcription initiation site (+ I)
could be determined by the specific nucleotide sequences around the + 1 site. Figure 3 shows the results
of S 1 nuclease assay using the mutated promoters.
The mutation of the . + 2 nucleotide does not show
any remarka ble change of the tran sc nptton actIvIty.
Only th e T ~C mutation gives some reduction in transc ription activity (60% decrease in activity compared
to wild type). The tra nscription initi a tion site, howeve r,
moves to - I site in the case of T ~C and T ~G
mutation s. Mutation of T ~A does not show a ny effects on transcription activity or its initiation site.
In the cases of T ~C and T ~G muta tion, the initiation site was moved to C residue of - 1. The reason
of this changed initiation site is not clea r, but we can
see th a t the residue at which the transcription starts
is followed by the T/A base pairing. This strongly
suggests that the tra nscription could be initiated at
the nucleotide just before the A!f base pairing.
3' Boundary oj the promoter
We have changed the sequence from + 12 to + 17
to form a BssHII restriction site, cha nging 5 out of
6 nucleotides in tha t region. At the sa me time nucleo-
Vol. 3 (1993)
Hyuk Ran Kwon ef al.
2345678M
-
45 base (-5)
11
-
40 base (+ 1)
TC
Figure 4. Transcription signal from the templates which have
one nucleotide change at + 2 site and Bss HII site from + 12
to + 17. Lane M, size marker of 45 bp; lanes I and 5, lanes
2 and 6. lanes 3 and 7, and lanes 4 and 8 are the same
in Figure 3 except that these templates have extra mutation
from + 12 to + 17 (BssHII site).
M
C- Ss sH
40 5
.1
.10
!
I
'20
I
."I
Transcription
initiation si te
AG TAliT TIiGGAGGTAC TTCATCCGAAAGCAliT TGAAGACAAGT Tc9tcl89acc£!£i!.S
"
100
ACT AC TTGGGAGG TACT TCA£CCGAAAGCAGTTGAAGACMC TTc9 t caagacc£!£9!i
·1
40
AG'AC TTGGGA GGTAC1TCAQGCCA.AAG CAGT TGAAGACAACTTcgtcaagaccill9!i
·1
110
ACT ACTTGGGAGGTACT TCAaGCGAAAGCAG TTGMGACAAGT Tcgt C18g8CCilli!i
.1
100
"'GT"'G T TGG GAGGT A C TT C"'T GCGMAGC"'G~CACAAGTTcgt C . lIgIICC~
.1
10
"'GT "' CTTGGGAGGT"'C1 T C"'£G C GAA"' GC AG~GACAAGT Tc gt c8a g .cc~
·1
A CT "'GTT GGGAGGT "' CTTC"'§CCGAAACCA C ~GACAA cT ' c gt r; il ilgacc~
-1
10
.1
100
AC TACTTCCCACC ' AC T 'CA~CCGAAACCAC~GAC.AA Cnc gt c ilagar; c~
lS Z/15
X of
transcriptlor'l
AC TACTTCCGA CCTAC T TCA T £C£.AAA£C£Cf...
CCAAGA C.AACTTr;gt caa gacc~
Not determined
Figure 6. Summary of assays of promoter mutations. Asterisks (*) show the transcription irt;tiation site and underlines
depict the mutated sequences.
2
Transcription signal
Figure 5. Transcription activity of promoter which has muta-
tions between + 3 to + 14. A separate end labeled probe
was made for each promoter, and all probes were labeled
to the same specific activity. Lane M. maker DNA (pBR322
DNA digested with MspI); lane 1, wild type promoter; lane
2. promoter contains the mutations between + 3 to + 14.
tions. These alterations did change the site of transcription initiation exactly same as the case of mutation
at + 2 site only (Fig. 6). This result shows that the
region from + 13 to + 17 is participated in the promoter activity, not in determining the transcription initiation site. At this time we can not explain why the
A-BssH mutation does not give any effects on the
transcription activity.
The plasmid which has 16 bp linker DNA at + 25
was proved to be actively transcribed in vitro like as
in vivo (Schultz et al., 1991; Choe et aI., 1992). Thus
we think that the linker insertion · at + 25 is probably
outside the 3' boundary of the yeast ribosomal gene
promoter. In the process of making a G-free cassette
for the ribosomal gene promoter using SrnaI restriction enzyme we substituted guanine nucleotides to cytosine up to + 14 (LS +2/ + 15, Fig. 2). These mutation completely kill the promoter activity (Fig. 5).
From the results of Figure 4 and 5, we conclude that
the 3' boundary of the promoter probably lies between
+ 13 and + 17.
Acknowledgment
This work was supported by a Genetic Engineering
Research grant from Ministry of Education.
References
tide +2 was changed from T to C, G , or A (our primary aim was to eliminate all T residues from the
immediate 5' end of the transcript so that we could
make a T-free cassette for this promoter region). These
combined alterations cause drastic change in promoter
activity (Figs. 4 and 6). In every case, except the template containing the A-BssH mutation, the transcription
activities were reduced to approximately 10% compared to the plasmids which do not have BssH muta-
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Yeast Ribosomal RNA Transcription
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