Download LETTERS Transcription and Translation are

LETTERS
Transcription and Translation are Coupled in Archaea
Sarah L. French,* Thomas J. Santangelo, Ann L. Beyer,* and John N. Reeve *Department of Microbiology, University of Virginia Health System; Department of Microbiology, The Ohio State University
Polysomes have been visualized by electron microscopy attached directly to dispersed strands of genomic DNA extruded
from lysed cells of the hyperthermophilic archaeon Thermococcus kodakaraensis. These complexes are consistent with
transcription and translation being coupled in this Archaeon, with translation of transcripts being initiated before the transcript is complete.
The presence or absence of a nuclear membrane has
historically been used as a taxonomic feature to divide
all life into 2 groups, eukaryotes or prokaryotes, respectively. However, there is now very substantial molecular
support for 3 primary phylogenetic domains (Woese
2000), and the classical definition of a prokaryote applies
to both Bacteria and Archaea. Given that Bacteria and Archaea do not constitute one coherent phylogenetic lineage,
that the definition based on the absence of a feature is scientifically invalid, and that prokaryotic is often inaccurately
used as meaning bacterial, Pace (2006) recommended that
the term prokaryote no longer be used.
In response, Martin and Koonin (2006) pointed out that
the absence of a nuclear membrane does provide the opportunity for ribosomes to bind to a transcript and so initiate
translation before the transcript is complete, and that this
could be a positive feature of being a prokaryote. Coupling
of transcription and translation has been documented and
studied extensively in Bacteria, where it provides the molecular basis for regulation of gene expression by attenuation
(Grundy and Henkin 2006). The close association of translating ribosomes with the advancing bacterial RNA polymerase can affect the structure of the growing transcript and so
has the potential to influence intrinsic transcription termination (Pan and Sosnik 2006) and can also prevent loading of
the transcription termination factor rho (q). In Bacteria, when
translation by pursuing ribosomes is halted or significantly
delayed, continued transcription is usually limited resulting
in the polarity observed in expression of promoter-distal
genes in multigene operons (Yanofsky 2003).
It seems reasonable to predict that Archaea will also
have exploited the regulatory opportunities offered by coupling transcription and translation, but there is no published
experimental support for this prediction. Archaeal genome
sequences and northern blot results do argue convincingly
that many archaeal genes are cotranscribed as members of
multigene operons, but now with over 30 archaeal genomes
sequenced only one putative attenuator has been identified,
and this seems likely to be regulated by a riboswitch rather
than by a translating ribosome (Rodionov et al 2003). Furthermore, archaeal genomes do not encode detectable homologues of q nor have functionally equivalent termination
factors been identified, and polarity has not been demonKey words: Archaea, prokaryote, coupled transcription, polysome,
electron microscopy.
E-mail: [email protected].
Mol. Biol. Evol. 24(4):893–895. 2007
doi:10.1093/molbev/msm007
Advance Access publication January 20, 2007
Ó The Author 2007. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
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strated in Archaea. Furthermore, although archaeal RNA
polymerases do exhibit intrinsic termination, they terminate
in response to DNA template sequences without an apparent requirement for nascent transcript folding (Santangelo
and Reeve 2006).
Given the importance of coupled transcription and
translation in Bacteria, and the surprising lack of evidence
for such coupling in Archaea, we repeated the classic chromatin spreading experiment of Miller et al. (1970) using
archaeal cells of the marine hyperthermophile Thermococcus kodakaraensis (Atomi et al. 2004). As shown in figure
1a, T. kodakaraensis cells are quasi-spherical and multiflagellate, and they lyse spontaneously when resuspended in
water. Electron microscopic examination of chromatin extruded from such cells clearly revealed the presence of polysomes containing up to 20 adjacent ribosomes attached
directly to dispersed strands of the archaeal genomic
DNA (figs. 1b–d). The polysome patterns were consistent
with the sequential direct binding of ribosomes to nascent
mRNAs. Polysomes were only rarely seen separated from
DNA. Given these observations, translation of mRNAs is
apparently initiated in T. kodakarensis before the transcript
is complete, and it seems likely that this is also the case in
most if not all Archaea. In this regard, it is noteworthy that
many but not all archaeal genes are preceded by sequences
consistent with ribosome-binding sites, but that some archaeal transcripts have no leader sequence (Torarinsson
et al. 2005). When and how ribosomes attach to these transcripts is clearly a puzzle, and experiments are also now
needed to determine if the presence of a translating ribosome has any influence on transcription termination by
the advancing archaeal RNA polymerase.
Given the results obtained, prokaryotes could be defined as species in which translation of transcripts can be
initiated before the transcript is complete (Martin and
Koonin 2006). Although this does define prokaryotes
positively at an organizational grade, it provides no clear
support for any particular phylogenetic relationship of prokaryotes to eukaryotes.
Methods
Aliquots (1 ml) were removed from cultures of T.
kodakaraensis strain KW128 growing exponentially in rich
media (MA-YT-S°) at 82 °C (Sato et al 2005). The time
between sampling and cell lysis was minimized by rapid
sedimentation (microfuge centrifugation for 7 s) and immediate resuspension in water or in 0.025% Triton X100 (pH
9). Samples of the resuspended cells were centrifuged onto
carbon-coated 300-mesh copper electron microscopy grids,
894 French et al.
FIG. 1.—Electron micrographs of genomic DNA extruded from lysed Thermococcus kodakaraensis cells. (a) A lysed cell showing flagella and
the spontaneous spreading of extruded chromatin. (b, d, and c) Dispersed strands of genomic DNA with polysomes attached shown at increasing
magnifications (bar 5 0.2 microns). In (d), the DNA strand is highlighted by yellow shading.
stained for 30 s using 1% ethanolic phosphotungstic acid,
washed with ethanol, stained for 1 min with 1% ethanolic
uranyl acetate, washed with ethanol, and air dried (Osheim
and Beyer 1989). Electron microscopy was carried out using a JEOL 100CX electron microscope.
Acknowledgments
This research was supported by Department of Energy
grant DE-FG02-87ER13731 (to J.N.R.), National Institutes
of Health (NIH) grants GM53185 (to J.N.R.) and GM63952
(to A.L.B.), and NIH fellowship 1F32-GM073336-01 (to
T.J.S.).
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William Martin, Associate Editor
Accepted January 3, 2007