JOURNAL OF BACTERIOLOGY, July 1995, p. 3897–3903 Vol. 177, No. 14
0021-9193/95/$04.00ϩ0
Copyright 1995, American Society for Microbiology
MINIREVIEW
Prokaryotic Introns and Inteins: a Panoply of Form and Function
MARLENE BELFORT, MARY E. REABAN, TIMOTHY COETZEE,† AND JACOB Z. DALGAARD‡
Molecular Genetics Program, Wadsworth Center and School of Public Health, State University of New York at Albany,
New York State Department of Health, P.O. Box 22002, Albany, New York 12201-2002
INTRODUCTION
Following their initial discovery, introns and RNA splicing
were considered, along with the nuclear envelope, as characteristics
that distinguish eukaryotes from prokaryotes (the eubacteria,
referred to simply as bacteria, and the archaebacteria,
referred to as archaea). This dogma became shaky with the
identification of putative introns in tRNA genes of archaea
(35) and finally crumbled with the discovery of self-splicing
group I introns in the phages of purple bacteria (9), which was
followed by the discovery of group I (40, 59, 72) and group II
introns (24) in bacterial cells. Another breakthrough was the
recent discovery of a nuclear pre-mRNA-like intron in a bacterial
plasmid (48). The variety of intervening sequences
(IVSs) that span the phylogenetic spectrum was further extended
by the discovery of inteins, elements that can splice at
the protein level, in eukaryotes (36), archaea (56, 71), and
bacteria (19, 20). This minireview will illustrate that interrupted
genes can no longer be considered the province of
eukaryotes, but rather that many forms of IVSs are phylogenetically
diverse, occurring also in bacterial and archaeal genomes
(Fig. 1).
GROUP I INTRONS
The first intron to be discovered in a prokaryote was a group
I intron, located in the thymidylate synthase gene of bacteriophage
T4 (9). Subsequently, group I introns were found in
other T4 coliphage genes (29) as well as in B. subtilis phage
genes (1, 28) and in tRNA genes of cyanobacteria and proteobacteria
(40, 59, 72). Additionally, group I introns were
found in the genomes of both chloroplasts and mitochondria,
which are endosymbiotic descendents of the cyanobacteria and
proteobacteria, respectively (reviewed in reference 3). As with
the canonical group I intron of Tetrahymena thermophila (8),
many of these introns can self-splice in vitro in the absence of
proteins.
Group I introns are spliced from the precursor RNA by a
series of transesterification reactions initiated by an exogenous
guanosine cofactor, resulting in ligated exons and a free intron
bearing the initiating guanosine at its 5Ј end (Fig. 2A) (reviewed
in references 8 and 60). This latter feature of the
reaction has been widely exploited to identify group I introns:
bacterial RNA extracts were incubated with [␣-32
P]GTP and
then screened for radiolabeled introns (29). It is therefore not
surprising that the majority of phage and eubacterial group I
introns discovered thus far have been shown to be self-splicing
in vitro. Nevertheless, it appears that certain proteins that bind
RNA nonspecifically, such as the E. coli ribosomal protein S12,
may provide chaperone-like activity to promote proper folding
of the RNA (11). This contrasts with the many fungal mitochondrial
group I introns that require specific protein splicing
factors, some encoded by nuclear genes, others (maturases)
encoded by the introns themselves (7, 42).
Despite a lack of primary sequence conservation, group I
introns share common short- and long-range pairings, P1
through P9 (Fig. 2A), which form the basis of a conserved,
catalytically active tertiary structure (49, 51). In addition, many
group I introns have open reading frames (ORFs) looped out
of these conserved structures (reviewed in reference 53). Several
of these ORFs encode endonucleases which play a role in
intron mobility, as is described below.
GROUP II INTRONS
Group II introns have been recently identified in cyanobacteria
(Calothrix spp.) and proteobacteria (Azotobacter vinelandii),
the respective progenitors of chloroplasts and mitochondria
(24), and in a bacterium close to the hearts of many,
Escherichia coli (23, 39). These discoveries are particularly
intriguing from an evolutionary standpoint, for group II introns
had previously been known to exist only in chloroplasts
and mitochondria of fungi and plants and because the group II
splicing mechanism is similar to that of nuclear pre-mRNA
introns. Indeed, it is widely held that group II introns served as
the ancestors of nuclear pre-mRNA introns (62).
As with nuclear pre-mRNA splicing, group II splicing initiates
when the 2Ј OH of a bulged nucleotide attacks the 5Ј
splice site, resulting in formation of a 2Ј-5Ј bond and a lariat
structure (Fig. 2B). Subsequently, the terminal OH of the 5Ј
exon attacks the 3Ј-splice site, resulting in ligation of the exons
and release of the intron lariat (reviewed in references 8 and
60). Like group I introns, group II introns adopt a conserved
secondary structure that is essential for splicing (50). This
structure consists of six conserved helices that emanate from a
central wheel (Fig. 2B). Sequence conservation is confined to
short strings of intron residues and the bulged A in domain VI
that forms the lariat branch site.
Although some group II introns can self-splice, they do so
only under nonphysiological conditions, and many require protein
cofactors, some of which are encoded by the intron (reviewed
in reference 42). The products of these internal ORFs
can act as maturases to facilitate splicing or as reverse transcriptases
(RTs) to promote group II intron mobility (41, 60).
A NUCLEAR PRE-mRNA-LIKE INTRON
An Ri plasmid gene of Agrobacterium rhizogenes has been
found to contain an intron in its untranslated leader region.
† Present address: Brain and Development Research Center, University
of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7250.
‡ Present address: Frederick Cancer Research and Development
Center, P.O. Box B, Frederick, MD 21702-1201.
3897
Surprisingly, this intron has the characteristic features of a
plant nuclear pre-mRNA intron: the dinucleotides GT at the 5Ј
end and AG at the 3Ј end of the intron, a T-rich region
preceding the 3Ј splice site, the appropriate AT content, and
homology to the plant consensus sequence at the 5Ј exonintron
junction (48). Transcription and splicing take place in
transformed Arabidopsis thaliana plant cells, and it is therefore
assumed that the plant spliceosomal machinery effects processing
of the bacterial pre-mRNA.
ARCHAEAL INTRONS
A single class of introns, unrelated to any of the aforementioned
ones, has been described in the archaeal kingdom.
Unique to this phylogenetic domain, they have been found in
tRNA genes of halophiles and in tRNA and rRNA genes of
hyperthermophiles (18, 25). Archaeal introns vary in size upwards
of 15 nucleotides, are catalytically inert, and share a
structural motif only at the intron-exon boundaries (38, 64, 65)
(Fig. 2C). This motif, a bulge-helix-bulge structure, is recognized
by a protein enzyme, an RNA endoribonuclease, that
excises the intron from the precursor RNA by cleavage in each
of the two bulges. The cleavage event creates 2Ј-3Ј cyclic phosphates
and 5Ј hydroxyl termini, a characteristic of nuclear
tRNA introns of eukaryotes. A poorly defined ligation reaction
creates a normal 5Ј-3Ј phosphodiester bond joining the exons
(37, 38, 64). Several archaeal introns possess ORFs, some of
which encode mobility-type endonucleases (16, 17, 47). Remarkably,
these archaeal enzymes contain the LAGLIDADG
amino acid motif, a conserved feature of DNA endonucleases
encoded by eukaryotic group I introns and by inteins (reviewed
in reference 53).
OTHER RNA INTERRUPTIONS
The 23S and 16S rRNA genes of diverse bacterial species
contain small (100 to 200 bp) IVSs, which are excised from the
RNA without exon ligation, resulting in fragmented rRNAs (6,
44, 66). Analogous bacterial 23S rRNA IVSs that contain
ORFs of 121 to 133 amino acids have also been found (58).
The ability of these fragmented rRNAs to assemble into an
active conformation appears to obviate the need for exon ligation.
These IVSs have the potential to fold into similar
stem-loop structures; the Salmonella IVS has been shown to be
excised by RNAse III, which has a preference for such duplex
conformations (6). The sporadic distribution of these sequences
among closely related organisms, and even among the
multiple rRNA gene copies of the same organism, plus the
presence of ORFs in some, suggest that these IVSs too are
mobile, having invaded rRNA genes.
Yet another type of IVS is found in bacteriophage T4, in
gene 60, encoding a topoisomerase subunit (32, 68). This 50-nt,
atypical IVS is neither spliced nor excised. Instead, it is bypassed
during translation by a gigantic frameshift event. Several
features have been identified as being required for ribosomal
hopping: a cis-acting 16-amino-acid segment of the
nascent peptide, matching ‘‘takeoff’’ and ‘‘landing’’ codons optimally
separated by 50 nt, a stem-loop structure at the takeoff
site, and a stop codon immediately following the takeoff site.
Ribosomal protein L9 also appears to play a role in the process
(30). The E. coli trpR gene also appears to produce a minor
product via a frameshift event that bypasses 55 nt (5). However,
the mechanism of the trpR frameshifting event differs
from that of T4 gene 60: there is no requirement for matching
FIG. 1. Phylogenetic distribution of introns and inteins. The distribution of introns and inteins is shown on a phylogenetic tree of the three primary kingdoms
(modified from reference 69). IVS types represented in circles are as follows: GI, group I; GII, group II; Ar, archaeal; In, intein. Those IVSs represented by filled circles
are nuclear, while the others are prokaryotic or organellar. The group I introns in gram-positive bacteria are in phage genomes. See reference 46 for the flagellate
mitochondrial group I intron; see text for other references.
3898 MINIREVIEW J. BACTERIOL.
FIG. 2. Splicing pathways and requisite structures. (A) Group I introns. Step 1, nucleophilic attack at the 5Ј splice site by the 3Ј OH of an exogenous guanosine
cofactor. Step 2, attack at the 3Ј splice site by the 3Ј OH of the 5Ј exon, resulting in exon ligation and release of the intron. Step 3, a third, optional, transesterification
reaction between the 3Ј OH of the intron and an internal site can result in cyclization. Exons are represented by boxes, and introns are represented by lines. The
secondary structure is shown below, with exons depicted as boxes and splice sites indicated by arrowheads. (B) Group II introns. Step 1, attack at the 5Ј splice site by
the 2Ј OH of an adenosine near the 3Ј end of the intron results in formation of a 2Ј-5Ј bond. Step 2, the 3Ј OH of the 5Ј exon attacks the 3Ј splice site resulting in exon
ligation and release of the intron lariat. The secondary structure is shown below, with conserved pairings designated I through VI, and with the bulged A in helix VI.
(C) Archaeal introns. Step 1, endonucleolytic cleavage at the bulge-helix-bulge motif, creating 2Ј,3Ј cyclic phosphates (circles). Step 2, exon ligation and optional
cyclization. (D) Inteins. Conserved amino acids are indicated at the junction of inteins (light shading) and exteins (dark shading). Step 1, protein splicing is initiated
either by nucleophilic attack on the carbonyl group of the upstream splice junction by the serine hydroxyl at the downstream junction or by an N-O acyl shift in the
upstream serine, followed by attack and transesterification by the downstream serine. Cysteine and threonine could replace serine in any of these reactions. Both
pathways yield a branched intermediate containing an alkali-labile ester bond linking the N-extein to the intein-C-extein. Step 2, the intein is freed by cyclization of
the conserved asparagine to form succinamide, and the ester linkage between the two exteins undergoes an O-N acyl shift, producing a standard linkage between the
two exteins (70).
VOL. 177, 1995 MINIREVIEW 3899
takeoff and landing codons or for a stop codon, and ribosomal
hopping is less efficient by an order of magnitude.
INTEINS—PROTEIN SPLICING ELEMENTS
Another dogma-breaking discovery was of genes which contain
in-frame inserts that are removed at the protein, rather
than at the RNA, level. The intervening polypeptide (intein) is
excised precisely from the precursor protein, and the flanking
polypeptides (exteins) are ligated to form the mature protein
(12, 14, 57). Inteins are widespread (Fig. 1), having been found
in the nuclear genes for a vacuolar ATPase subunit in the
eukaryotes Saccharomyces cerevisiae and Candida tropicalis, in
the DNA polymerase genes of the archaea Thermococcus litoralis
and Pyrococcus sp. strain GB-D, and in the recA genes of
the eubacteria Mycobacterium leprae and M. tuberculosis (compiled
in reference 57).
There are several conserved amino acids at the intein splice
junctions, including a histidine-asparagine pair at the C terminus
of the intein and serine, cysteine, or threonine residues
downstream of both splice junctions (compiled in reference
71). Several models for the molecular mechanism of protein
splicing have been proposed (13, 67, 70, 71), but the details
have not yet been resolved. Figure 2D shows the most recent
model, in which an alkali-labile branched intermediate is resolved
by cyclization of the C-terminal asparagine of the intein
(71). In addition to these remarkable properties, all known
inteins contain the LAGLIDADG motifs characteristic of
homing endonucleases, and some have been shown to possess
analogous nucleolytic activities (53).
INTRON AND INTEIN MOBILITY
Some of the group I intron ORFs have been found to encode
double-stranded DNA endonucleases, which recognize and
cleave a cognate intronless allele at or near the site of intron
insertion. It has been shown for several of the phage and fungal
mitochondrial introns that repair of this double-strand break
results in the incorporation of the intron, including its ORF
(Fig. 3A). Thus, the ORF confers mobility on its host intron.
This process is referred to as homing, and the endonucleases
are referred to as homing endonucleases (reviewed in reference
41). Homing is a highly efficient gene conversion event
and involves coconversion of the adjacent exon markers, which
results from exonucleolytic degradation of the cleaved recipient.
Interestingly, the S. cerevisiae vacuolar ATPase intein
ORF has been shown to home into inteinless alleles by the
same pathway, initiated by the intein’s endonuclease activity
(26).
Although endonucleases with LAGLIDADG motifs similar
to those in the eukaryotic homing endonucleases and inteins
have been found in archaeal introns, homing remains to be
demonstrated in this system. Nevertheless, the discovery of an
endonuclease ORF related to the group I intron ORFs in a
different type of intron (16) fuelled arguments that the endonuclease
ORF is the true mobile element (41). By finding
refuge in a splicing element, host viability would be maintained,
while the intron acquires mobile properties. This scenario
has been further supported by the demonstration that
endonuclease recognition and cleavage sites lie hidden within
some introns. The occurrence of such sites which are split by
the ORF lends credence to the hypothesis that the endonuclease-ORF
invaded the intron as the primary agent of mobility
(45).
Mobility of group II introns is mechanistically different from
that of group I introns (Fig. 3B). These introns are capable of
homing (43, 52) as well as transposition reactions (54, 61), both
of which appear to be mediated by the conserved intron-encoded
RTs. Group II intron homing, which thus far has been
demonstrated only for yeast mitochondrial introns, is still a
poorly defined process. While homing involves coconversion of
flanking exon markers, implicating exon sequences in the mobility
event, the process has a remarkable requirement for
splicing proficiency of the intron. Thus, although intron cDNA
for homing is likely to be derived from pre-mRNA, splicing or
a competent intron structure is needed. Two proposals advanced
for this requirement are as follows. (i) An RNA structure
may be required for cDNA synthesis by RT (52). (ii)
There may be a role for the intron in endonuclease function
required in the cDNA integration step (4, 52).
Recent evidence favors group II intron transposition to nonallelic
sites through RNA intermediates (54, 61). The most
likely pathway is via reverse splicing into heterologous RNA.
Subsequent reverse transcription and homologous recombination
into the heterologous site would fix the intron at the
nonallelic locus on the genome (Fig. 3C). Theoretically, group
I introns could move by a similar mechanism. Although no
group I introns are known to contain cognate RTs, and no
complete transposition events have yet been reported, the selfsplicing
group I intron in the Chlamydomonas reinhardtii chloroplast
23S rRNA gene has been found to undergo the first
step of reverse splicing into the host cytoplasmic 5.8S rRNA
(63).
Although the self-propelled mobility pathways of group II
introns have not yet been observed in bacteria, the occurrence
of RT-ORFs in these introns raises expectations of their mobility.
Furthermore, like the RT-containing bacterial retrons,
which have been discovered both in freestanding form and in
prophages (34), some group II introns are contained within
DNAs that have the hallmarks of mobile elements (23, 39).
These findings suggest that group II introns have superimposed
mobility mechanisms. They have the potential to home
or transpose by virtue of their built-in RTs, while mobility may
also be conferred by the putative transposition ability of the
elements in which they reside.
INTRONS AND INTEINS—THE WHYS
AND WHEREFORES
Why are introns and inteins present at all in prokaryotes?
The streamlined nature of prokaryotic genomes, with their
pressure to shed excess DNA to effect rapid replication, is a
major force at work to eliminate them. Furthermore, the physical
constraints of the prokaryotic environment may be restrictive:
uninfected, wild-type E. coli cells have been shown to lack
the requisite accessory functions for homing (10), and coupling
of transcription and translation may interfere with splicing,
thus decreasing the tolerance of introns. Finally, opportunities
for dissemination are limited in prokaryotes, compared with
eukaryotes, which enjoy efficient sexual reproduction (31, 33).
Countering these eradicative pressures is the sheer invasiveness
of these elements. Introns and inteins can be considered
selfish DNAs, and, as has been proposed for transposable
elements and nuclear mRNA introns (21, 55), may survive by
their ability to invade and multiply without significantly compromising
their host. In fact, inteins would represent the ultimate
parasites, having the self-contained ability to invade a
gene, independent of the splicing function of a host intron. The
fact that homing has evolved numerous times independently
for different kinds of IVSs suggest that mobility is a strong
evolutionary driving force and that their parasitic nature wins
out over forces to eliminate them. As an additional incentive to
3900 MINIREVIEW J. BACTERIOL.
retain them, the host organism may find some aspect of the
intron/intein beneficial, or the IVS may evolve properties beneficial
to their host and/or themselves (2, 15).
A popular theory is that the self-splicing introns are molecular
fossils from the primitive RNA world. This hypothesis is
fueled by demonstrations that group I introns can function as
replicases (22) and is consistent with the presence of group I
and group II introns in bacteria. These introns, specifically the
group II introns, are postulated to have given rise to the nuclear
pre-mRNA introns prevalent in modern metazoans (62).
The discovery of a nuclear pre-mRNA-like intron in an
agrobacterium (48) provides a provocative conceptual bridge
between the bacterial self-splicing group II introns and the
spliceosome-dependent pre-mRNA introns in eukaryotes.
Although neither the catalytic group I and group II introns
nor the pre-mRNA introns have yet been found in archaea,
archaeal tRNA introns are similar in both position and splicing
pathway to tRNA introns of eukaryotes (reviewed in reference
41). It is conceivable that the relatively close phylogenetic
relationship with the archaea may explain the source of tRNA
introns in eukarya. These speculations on the origins of eukaryotic
nuclear pre-mRNA and tRNA introns are consistent
with the postulated chimeric nature of eukarya, as organisms
which arose from a fusion between members of the bacterial
FIG. 3. Intron mobility pathways. Wavy lines, RNA; straight lines, DNA; thick lines, introns; thin lines, exons. (A) Group I intron and intein homing through repair
of double-stranded DNA breaks. Endonuclease-mediated cleavage of the recipient alleles stimulates a gene conversion event that results in intron or intein inheritance
in a DNA-based pathway. (B) Group II intron homing through an RT-mediated pathway. Here, pre-mRNA is postulated to act as a template for cDNA synthesis by
RT. The role of intron structure or splicing (dashed line) and the mechanism of site-specific integration of the cDNA remain conjectural. (C) Intron transposition by
reverse splicing. Although reverse splicing is depicted here with RNA as the recipient, it is also mechanistically possible for DNA to be the recipient. The cDNA is then
proposed to act as a recombination substrate with genomic DNA in double-stranded (as shown) or single-stranded form. (Adapted from reference 4, with permission
from the publisher.)
VOL. 177, 1995 MINIREVIEW 3901
and archaeal kingdoms (27). Thus, introns are not only of
functional interest to the molecular biologist, but their occurrence
and properties continue to feed into theories of molecular
evolution and the origins of biological diversity.
ACKNOWLEDGMENTS
We are thankful to Maryellen Carl for manuscript preparation,
Maureen Belisle for illustrations, and Mary Bryk, Vicky Derbyshire,
Joyce Huang, Monica Parker, and Elisabeth Tillier for comments on
the manuscript.
Work in the laboratory of M.B. is funded by NIH grants GM39422
and GM44844. M.E.R. is supported by NIH postdoctoral grant
GM14915.
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