Title:
NON-CANONICAL TRANSCRIPTION INITIATION IN BACTERIA
Authors: Natalya Panova1, Ivan Barvík2, and Libor Krásný1†
Affiliations:
1
Institute of Microbiology, Czech Academy of Sciences, v.v.i., Vídeňská 1083, 142 20 Prague 4,
Czech Republic.
2
Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke
Karlovu 5, 121 16 Prague 2, Czech Republic.
†
Corresponding author. E-mail: krasny@biomed.cas.cz
One sentence summary:
NAD can be used by bacterial RNA polymerase as a substrate to prime transcription.
ABSTRACT
Recently, nicotinamide adenine dinucleotide (NAD) was identified in some prokaryotic RNAs at
their very 5’ ends, reminiscent of the posttranscriptionally attached 7-methyl guanosine (the cap)
in eukaryotic mRNA. The eukaryotic cap serves a number of vital functions, including effects on
RNA stability and translational control. The mechanism of attachment of NAD to bacterial
RNAs has been enigmatic so far. Here we demonstrate that it is incorporated by RNA
polymerase (RNAP) that can use this non-canonical nucleotide to prime transcription in a
promoter-dependent manner. In silico modeling provides structural insights that reveal the
positioning of NAD in the active site during transcription initiation. Thus, bacterial RNAP can
utilize a wider variety of substrates and creates more diverse transcriptomes than previously
believed.
MAIN TEXT
In eukaryotic cells 7-methyl guanosine (the cap; Fig. 1A) is attached by the capping enzyme
complex to the 5’ end of the nascent pre-mRNA as it emerges from RNA polymerase II. It is
important for transcription, splicing, polyadenylation, and nuclear export of mRNA and U-rich,
capped snRNAs. Furthermore, the cap is required for efficient translation of the mRNAs (1).
Several years ago it was discovered that nicotinamide adenine dinucleotide (NAD; Fig 1B) was
present in total RNA in Escherichia coli (2). Recently, the identity of the RNAs that contain
NAD at their 5’ ends was identified (3). The RNA that was modified most frequently was RNAI,
which is crucial for the replication control of ColE1 type plasmids. Besides RNAI, a number of
other RNAs were identified, some of them important for stress response of the cell. It was shown
that the presence of NAD at the 5’ end increased the stability of the RNA in vitro (3). Thus,
NAD was dubbed as the prokaryotic cap. It had been originally speculated that NAD might be
incorporated into RNA by RNA polymerase (RNAP) but published experimental results argued
to the contrary (2). Hence, it was proposed that the joining of the cap to the RNA occurs
posttranscriptionally, perhaps by the enzymatic machinery that is normally involved in NAD
biosynthesis (4). Inconsistent with this proposal is the observation that phage T7 RNAP can
utilize NAD, coenzyme A (CoA), and flavin adenine dinucleotide (FAD) to initiate transcription
in vitro (5). However, as it is a single subunit enzyme, which is significantly less complex than
bacterial RNAPs (6), it may, or may not, function differently with respect to utilizing noncanonical initiating substrates than bacterial RNAPs. The latter is supported by the fact that
bacterial RNAP can use ribo-oligonucleotides (nanoRNAs) as primers for transcription both in
vitro and in vivo (7). NanoRNAs are typically 2-4 nucleotides (nt) long and their 3’ ends basepair with the +1 position of the template strand where transcription starts. The presence of the
extra 1-3 nt at the 5’ end appears not to hinder transcription initiation. Therefore, we
reinvestigated the possibility that bacterial RNAP may use NAD as a substrate.
We used E. coli RNAP as the enzyme and RNAI as our model RNA. RNAI is transcribed from a
70 (the main sigma factor in E. coli) dependent promoter that initiates transcription with ATP
(RNAIp; Fig. 2A) and is present in the transcription vector p770 (Fig. 2B) (8). We hypothesized
that the adenine moiety of NAD may base pair with the +1 position of the template strand and
thus initiate transcription. We conducted parallel in vitro multiple round transcription
experiments in which the radioactive label was either in UTP (incorporation throughout the
template) or NAD (incorporation at the 5’ end). Fig. 2C shows that both labeled compounds
were efficiently incorporated, consistent with the hypothesis that bacterial RNAP can use NAD
as a substrate.
To verify the results, we cloned RNAIp into a different locus of the p770 plasmid while we still
kept RNAI with its promoter at the native locus, serving as an internal control (Fig. 2B). We
cloned two variants of RNAIp: one initiating with ATP (+1A) and the other with GTP (+1G, Fig.
2AB). Fig. 2D shows that while with radioactive UTP the transcription was always efficient
regardless of the identity of the +1 nucleotide, with radioactive NAD the transcript was detected
only in the case of the +1A promoter variant. We then used various non-radioactive substrates as
competitors (Fig. 2E). NAD, NADH, AMP, and GTP were used in excess over radioactive NAD
and all of them with the exception of GTP were efficient competitors. Hence, only those
structures that contained adenine were able to prevent the incorporation of the radiolabeled
NAD.
We then tested whether NAD is incorporated into RNA with equal efficiency regardless of the
promoter sequence. We used Pveg as a second promoter. Pveg is a strong model promoter from
Bacillus subtilis that is well characterized and recognized also by E. coli RNAP (9). Fig. 2F
shows that the incorporation of NAD to transcripts originating from Pveg was achieved with
lower efficiency than to transcripts originating from RNAIp, suggesting that the sequence of the
promoter or/and the early transcribed region affect the ability of RNAP to prime transcription
with NAD. Further, it is possible that nudix phosphohydrolases (10) that break the
pyrophosphate bond and remove the cap from RNAs may display differential efficiency in the
cell towards different NAD-capped RNAs, thereby providing an additional layer in regulating
the presence of NAD at the 5’ ends of RNAs.
To provide spatial insight into transcription initiation with NAD we performed in silico modeling
based on experimentally determined 3D structure of bacterial RNAP that accepts two initiating
ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation (11).
Fig. 3 shows a transcription initiation complex with NAD and CTP in the active site of Thermus
thermophilus RNAP. The adenosine part of NAD occupies position +1 (which overlaps with the
RNA 3' end binding site in elongation complex). The nicotinamide part of NAD mimics a
nucleotide in the -1 position. Its carboxamide group is hydrogen bonded toward the cytosine
nucleobase of the template DNA strand in position -1. The ability of nicotinamide to bind
different nucleobases can be expected based on analogy with Ribavirin, which pairs equally well
with either uracil or cytosine depending on the rotation of its carboxamide moiety (12).
Furthermore, the 2’ hydroxyl group of the ribose attached to nicotinamide is hydrogen-bonded
with the cytosine nucleobase of the template DNA strand in position -2. Phosphate groups of
NAD are stabilized by contacts with side chains of Lys-838, Lys-846. These amino acids are
conserved in all cellular RNAPs (their counterparts in E.coli RNAP are Lys-1065, Lys-1073).
In conclusion, we show that bacterial RNAP can use NAD as a non-canonical substrate to prime
transcription. The NAD-capped RNAs are more stable than non-capped ones (3), which is
reminiscent of the role of the 5’ end phosphorylation status of RNAs that can affect the half-life
of RNA. When the RNA contains triphosphate at the 5’ end it provides a stabilizing effect
against RNAses that require the 5’ end to be monophosphorylated (13;14). The molecular
mechanism of the NAD-mediated stabilization of RNAs likely operates in the cell on a similar
basis. Considering the ability of bacterial RNAP to utilize NAD, it is possible that other
coenzymes, such as CoA and FAD, may be also used and thus bacterial RNAP may employ a
significantly wider set of initiating non-canonical substrates than previously believed; this in turn
is likely to specifically shape the transcriptional landscape and thus affect bacterial gene
expression. It is needed to establish under which conditions the NAD-capping is most
physiologically relevant.
Acknowledgements:
This work was supported by Czech Science Foundation Grants: 14-04289S
and 15-05228S (LK), and instrumental equipment provided by C4Sys infrastructure
was used.
Figure 1
A
B
Figure 1. Structures of eukaryotic and prokaryotic caps. A. 7-methyl guanosine attached to
adenosine via 5’ to 5’ triphosphate link. B. Nicotinamide adenosine dinucleotide.
Figure 2
A
B
C
NAD*
D
UTP*
TEST RNA: +1A
NAD*-TEST RNA
RNAI
NAD*-RNAI
E
p770
5.1 kb
+1G
NAD*
RNA1(+1A)wt
RNA1(+1G)
Pveg wt
-35
-10
+1
5’-TTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTG-3’
5’-TTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGGCAGTATTTG-3’
5’-TATTTGACAAAAATGGGCTCGTGTTGTACAATAAATGTAGTGAGGTGG-3’
Ori
RNAIp
-
NAD NADH AMP GTP
TEST RNA-UTP*
NAD*-RNAI
F
RNAI-UTP*
NAD*
0.5 1.0
2.0
Pveg
UTP*
1 mM competitor
UTP*
2.0
1.0
0.5
Pveg
RNAIp
RNAIp
Rel. transcription
Rel. transcription
Figure 2. Escherichia coli RNAP uses NAD to prime transcription. A. Sequence of promoters
and early transcribed regions (to +10) used in the study. -10, -35 hexamers and the transcription
start sites (+1) are indicated. We note that the sequence of RNAIp may somewhat differ between
different plasmids (15). B. A schematic view of the p770 plasmid. The relative positions of
RNAIp and the locus where various promoters can be cloned to transcribe the TEST RNA are
indicated with arrows. C. Multiple round transcriptions from RNAIp. The position of the
radioactive label is indicated with asterisk next to the respective compound. This and the
subsequent experiments were repeated at least three times. D. Multiple round transcriptions with
either radiolabeled NAD or UTP (indicated on the right hand side the gels). Each template
contained two promoters: (i) native RNAIp and RNAIp at a second locus. The second locus
RNAIp was either +1A or +1G (TEST RNA). The identity of the transcripts is indicated on the
left hand side of the gels. E. Multiple round transcriptions from RNAIp in the presence/absence
of various competitors. The label was in NAD. F. Multiple round transcriptions were carried out
from a plasmid template containing native RNAIp and Pveg. The identity of the chemical that
contained the radioactive label is indicated above the gels. Quantitation is shown next to each
gel. The error bars indicate ±SD. The activity of Pveg was set in both cases as 1.
Figure 3
-2
iNAD
-1
+1
+2
iCTP
Figure 3. De novo transcription initiation complex with NAD (replacing the first iNTP) and
iCTP in the active site of RNAP. Nucleic acids and amino acid side chains of Lys-838, Lys846 are shown as stick models, and the bridge helix (yellow), trigger loop (orange), DFDGD
motif (green), and sigma region 3.2 (magenta) are shown as cartoon models. Initiating NAD
(iNAD) binds at the active site of RNAP through multiple interactions, including base pairing
with the +1 DNA base, hydrogen bonding of the carboxamide group with the -1 DNA base
(dashed line), ribose-base interactions with the -2 DNA base (dashed line), and salt bridges with
conserved lysine side chains. iCTP is stabilized by aspartate residues of the DFDGD motif of the
' subunit coordinating the Mg2+ (green spheres).
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Supplementary Materials
NON-CANONICAL TRANSCRIPTION INITIATION IN BACTERIA
Natalya Panova1, Ivan Barvík2, and Libor Krásný1†
1
Institute of Microbiology, Czech Academy of Sciences, v.v.i., Vídeňská 1083, 142 20 Prague 4,
Czech Republic.
2
Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke
Karlovu 5, 121 16 Prague 2, Czech Republic.
†
Corresponding author. E-mail: krasny@biomed.cas.cz
CONTENTS:
MATERIALS AND METHODS
Promoter constructs
In vitro transcription
NAD, NADH, AMP, GTP titration
In silico modeling
SUPPLEMENTAL REFERENCES
Materials and Methods
Promoter constructs
Promoter constructs were created by aligning two complementary oligonucleotides (-38 to +10
for Pveg, and -39 to +10 for RNAI purchased from Eastport LifeScience, Czech Republic) and
cloned into p770 digested with EcoRI and HindIII. Upon transformation into E. coli DH5
clones with inserts were identified and verified by sequencing (promoter/plasmid code:
RNAIpwt/LK1680; RNAIp+1G/LK1700; Pveg/LK1679).
In vitro transcription
Multiple-round transcriptions were carried out in 40 mM TRIS-HCl buffer, pH 8.0 containing 10
mM MgCl2, 1mM DDT, 0.1 mg/ml BSA, and 90 mM KCl. Concentrations of the nucleotides
were as follows: 200 µM CTP, 100 µM GTP, 2 µM ATP, 10 µM UTP unless indicated
otherwise. Each reaction contained 100 ng of supercoiled plasmid DNA. Radioactive []32PUTP was purchased from Hartmann Analytic (Germany) and added at 0.034 µM (1 µCi/10 µl) .
When added, the final concentration of 32P-NAD was 0.6 µM (Hartmann Analytic, Germany).
The reaction mixtures were preincubated for 5 min at 37º C and transctiriptions were initiated
with E. coli RNA polymerase holoenzyme (Epicenter, USA) at a final concentration of 30 nM.
Reaction was allowed to proceed for 15 min at 37º C and was terminated with 10 µl of loading
buffer containing 95% formamide, 20 mM EDTA. The reaction products were separated by
electrophoresis on 7 M urea 7% polyacrylamide gel in 1xTBE (Tris-Borate-EDTA, pH 8.0)
buffer. The dried gels were scanned with Molecular Imager_FX (Bio-Rad). The amounts of
transcripts were quantified with QuantityOne software (Bio-Rad).
NAD, NADH, AMP, GTP titration
Multiple-round transcriptions were performed as described above. The only difference was the
presence of 1 mM NAD, NADH, AMP, or 1 mM GTP (purchased from Sigma).
In silico modeling
We performed in silico modeling of transcription initiation complex with NAD in the active site
of T. thermophilus RNAP based on the experimentally determined of X-ray structure (11) (amino
acids stabilizing both iNTPs are evolutionary conserved, i.e. all observations are directly
appliccable to E. coli RNAP). ATP in the active site of RNAP was replaced with NAD. The
TGC sequence in the template DNA strand facing NAD was replaced with TCC to match the
promoter used in our experiments. The transcription initiation complex was surrounded by water
molecules and the resulting simulated system was relaxed by short minimization and molecular
dynamics run produced by means of the NAMD software package (S1) using standard force
fields (i.e. 99SB (S2-3) for RNAP and nucleic acids, GAFF (S4) for NAD and TIP3P (S5) for
water molecules).
SUPPLEMENTAL REFERENCES
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Schulten K. Scalable molecular dynamics with NAMD. J Comput Chem. 26 (2005) 1781-802.
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Amber force fields and development of improved protein backbone parameters. Proteins 65 (2006)
712-25.
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the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers.
Biophys J. 92 (2007) 3817-29.
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amber force field. J Comput Chem. 25 (2004) 1157-74.
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