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412 LAMPSON

Figure 4. The unique structure of msDNA. msDNA is a small, abundant satellite DNA easily observed

by gel electrophoresis of DNA extracts. For example, N. exedens produces two different msDNAs

(inset photo) designated Ne165 and Ne144 based on the size of the DNA strand in each molecule. The

structure of msDNA-Ne144 is shown. This msDNA is composed of a single-strand of DNA containing

144 deoxyribonucleotides that folds into a long stable stem-loop structure. Joined to the 5’-end of the

DNA chain is a 72 ribonucleotide, single-stranded RNA molecule. The RNA strand is joined to the

5’-end of the DNA molecule via a 2’–5’ phosphodiester bond at a specific internal guanosine residue

(circled G) of the RNA molecule. In addition to stem-loop folding in the RNA chain, a small region of

base-pairing occurs between the very 3’-end of the DNA chain and the 3’-end of the RNA chain

PROKARYOTIC REVERSE TRANSCRIPTASES 413

Figure 5. msDNA is synthesized by reverse transcription. A smaller region of a longer mRNA transcript

of the retron msr-msd-ret operon will serve as both a primer and template for the synthesis of msDNA.

Folding of the template RNA (mediated by inverted repeats a1-a2 and b1-b2) is crucial to form the

2’-OH primer at a specific guanosine residue (circled G) to initiate cDNA synthesis by the retron RT.

After synthesis of cDNA (dashed line) is complete, part of the RNA template remains joined to the

5’-end of the cDNA molecule to yield the complete msDNA

3.4. msDNA is Synthesized by Reverse Transcription

The interesting way msDNA is reverse transcribed helps explain its unusual

structure. Synthesis of msDNA begins from an RNA transcript (of the msr-msd

genes) that folds into a specific secondary structure (Dhundale et al., 1987). The

2’-OH group of a specific guanosine (circled G, Fig. 5) within this folded RNA

414 LAMPSON

serves as a primer to initiate cDNA synthesis (Hsu et al., 1989). The retron RT

utilizes this 2’-OH group to incorporate the first deoxyribonucleotide using the

folded RNA as a template. In close concert with the extension of the cDNA strand is

removal of the lagging RNA template by an RNase H activity apparently provided

by the host cell (Lampson et al., 1989a; Shimamoto et al., 1995). Host RNase H

plays a similar role in removal of the RNA template in group II intron retro-mobility

(Smith et al., 2005). At a specific location on the RNA template, cDNA polymer-

ization stops, resulting in the msDNA molecule observed in cell extracts. That is,

a short cDNA chain covalently joined to the remainder of the RNA molecule that

served as a primer-template for its synthesis.

3.5. Retrons and the Host Cell

While group II introns occur quite frequently in bacterial genomes, retron elements

appear far less frequently and tend to occur sporadically within a population of the

same species. For example, among the 75 strains of E. coli that make up the ECOR

collection, all 75 strains contain at least one group II intron element and one strain,

ECOR 38, has as many as 15 copies of the same intron element in its genome

(Dai and Zimmerly, 2003). In contrast, only 11 of the 75 ECOR strains contains a

retron element and no report exists of a bacterial chromosome containing more than

one copy of the same retron element (Herzer et al., 1990). Nevertheless, retrons

are widely found among diverse taxonomic groups of the prokaryotes including

the alpha, beta, gamma, and delta proteobacteria, fusobacteria, Gram positives,

cyanobacteria, and one example from the archeon Methanosarcina acetivorans

(Rest and Mindell, 2003; Lampson et al., 2005).

Although it is not known if retrons are mobile DNAs, an increase in the frequency

of spontaneous mutations is observed when thousands of copies of the msDNA

molecule are produced during over-expression of a retron in E. coli (Maas et al.,

1994, 1996). Here, an increased frequency of frame-shift mutations occurred due

to binding and titrating out of the host repair protein MutS to mis-matched base

pairs in msDNA. In another interesting observation, numerous partial copies of

the msDNA sequence (i.e. the msd gene) were detected in a bacterial chromosome

(Lampson and Rice, 1997; Lampson et al., 2002). Evidence suggested that these

repeated DNA sequences could be retrotransposed copies of msDNA produced by

the corresponding retron RT.

4. DIVERSITY GENERATING ELEMENTS

The third type of prokaryotic RT containing element is in the recently described

diversity generating retroelements. These elements are so named because they

appear to function as a special mechanism to produce sequence variation in a phage

gene encoding a tail protein. By generating nucleotide substitutions at 23 sites in

a 134 base-pair VR region at the 3’ end of the phage gene, many variants of the

phage tail protein can be produced (Fig. 6) (Liu et al., 2002). By changing certain

amino acids at the C-terminal end of this tail protein, the phage can recognize and

PROKARYOTIC REVERSE TRANSCRIPTASES 415

Figure 6. Diversity generating elements (DGEs) produce DNA sequence variation by reverse

transcription. DGEs contain an ORF encoding a typical bacterial RT. Upstream of the RT-ORF is a

matching pair of directly repeated sequences designated VR and TR. Synthesis of a cDNA copy of the

TR sequence by the DGE RT eventually introduces nucleotide variation in the VR sequence. Because

the VR repeat sequence is part of an upstream ORF, this produces changes in the amino acid sequence

of the corresponding protein product (in this example variation occurs in a phage tail protein). See text

for more detail

bind to alternative receptor molecules on the surface of the bacterial host that it

infects. Thus, the presence of this diversity generating retroelement can confer a

direct selective advantage on the phage organism (Doulatov et al., 2004).

The diversity generating elements appear to be composed of a long ORF encoding

a typical RT similar to retroviral RT and especially analogous to the bacterial RTs.

The protein product of this ORF has been shown to be enzymatically active in a

standard RT assay (Liu et al., 2002). Upstream from the RT-ORF DNA is a pair

of 134-base, directly repeated sequences designated VR and TR. The TR sequence

is just 5’ to the start of the RT-ORF and is termed the template sequence. Further

upstream, the VR sequence is found as part of the extreme 3’ end of an upstream

ORF (Fig. 6). The VR sequence is termed the variable region because nucleotides

at 23 specific positions in this sequence will change among variants produced by

this element (Liu et al., 2002, 2004). With the TR region of a mRNA serving as a

template, sequence variation is thought to be produced by the reverse transcription

of a cDNA copy of the TR sequence (Fig. 6). This cDNA copy then inserts into

the VR region by what is termed a site-specific homing event (Doulatov et al.,

2004). Eventually the cDNA copy containing the nucleotide substitutions replaces

the original VR sequence. This, then produces amino acid changes in the phage tail

protein encoded by this upstream ORF.

Through sequence homology searches of sequenced microbial genomes, diversity

generating elements have so far been found in several diverse groups of bacteria

including Bordetella, Vibrio, Bacteroides, Treponema, and several cyanobacteria

(Doulatov et al., 2004).

5. APPLIED USES FOR PROKARYOTIC RTS

Currently, almost all commercially marketed RTs are eukaryotic enzymes. That is,

they are purified or cloned from animal retroviruses or are genetically engineered

derivatives of these enzymes. For example, the mutational inactivation of the

416 LAMPSON

C-terminal RNase H domain from the RT of Moloney murine leukemia virus

(M-MLV) yields an RT with greatly improved efficiency of cDNA synthesis in

vitro (Kotewicz et al., 1988). Retroviral RTs are today extensively used for many

applications that require synthesis of cDNA, from construction of cDNA libraries

to RT-PCR. However, the newly described prokaryotic RTs are a highly diverse

group of enzymes with novel properties and represent an emerging new tool with

potential new applications for RTs.

5.1. Targetrons

An example of a bacterial RT that is currently marketed as a genetic tool is the so

called targetron (Sigma-Aldrich). Targetrons take advantage of the way the group

II intron RT works in conjunction with intron RNA to mediate insertion of the

intron into a site on DNA (Guo et al., 2000). While the intron RT protein binds

and unwinds the target DNA molecule, it is specific sequences within the intron

RNA that recognize and base-pair with the homing site (insertion site) in DNA.

This allows for a very efficient and site-specific insertion of the intron into the

homing site on the target DNA molecule by the retrohoming mechanism. The

targetron system allows the user to reprogram the group II intron RNA molecule to

recognize a new insertion site in a DNA sequence of their choosing. This is done

by changing specific nucleotides in the intron RNA, using a mutagenic primer, so

that it will recognize and base pair to a new homing site (Guo et al., 2000). The

resulting targetron system is a highly efficient, site-specific insertion vector that

can insert and disrupt almost any desired bacterial gene in a cell where this group

II intron can be expressed (Frazier et al., 2003; Zhong et al., 2003). In addition,

by using a mutagenic primer-PCR reaction that incorporates random nucleotides,

the intron RNA can be reprogrammed to insert into many random sites in a target

chromosome. Such a randomized targetron vector was used to produce a gene

“knock-out” library in E. coli where most non-essential genes were shown to be

disrupted by an intron insertion (Yao et al., 2005).

5.2. Thermophilic RT

Recently, a group II intron was discovered in the thermophilic bacterium

Geobacillus stearothermophilus. The intron encoded protein was cloned, over-

expressed in E. coli and shown, like its host organism, to be heat stable. The

RT can reverse transcribe RNA at temperatures as high as 75



C (Vellore et al.,

2004). One of the big problems in using retroviral RTs to produce cDNA is that

the enzyme will prematurely pause during polymerization of DNA. This is due to

formation of secondary structures in the RNA template resulting in truncated cDNA

products (Harrison et al., 1998). The length of cDNAs produced by RT can be

greatly increased by carrying out reverse transcription at temperatures above 55



These hot temperatures will melt the secondary folding in the RNA template and

alleviate pausing of the RT (Fu and Stuve, 2003; Hawkins et al., 2003). Several

PROKARYOTIC REVERSE TRANSCRIPTASES 417

retroviral RTs have now been modified to increase their thermo-stability and activity

at higher temperatures for this reason. The discovery of a genuine heat-stable RT

from a thermophilic bacterium that remains active at higher temperatures could

also prove useful for synthesizing long cDNAs. A group II intron has also been

reported from the related thermophile G. kaustophilus (Chee and Takami, 2005).

This intron interrupts a functional recA gene and was shown to splice at temperatures

above 70



C in vivo.

5.3. msDNA

Although its function remains elusive, a number of interesting potential applications

for the retron-msDNA system have been explored. For example, msDNA can

be used as a type of antisense DNA to repress the expression of a particular

gene. In the stem-loop structure of msDNA the unpaired bases in the loop can

be greatly expanded to include more DNA without affecting the high level of

msDNA produced by the retron RT. Indeed, such an engineered retron was made

so that its msDNA contained a large loop of DNA sequence complementary to the

translation initiation region on the transcript for the lpp gene of E. coli (Mao et al.,

1995). The lpp gene codes for the highly abundant E. coli lipoprotein of the outer

membrane. Over-expression of this engineered retron caused a 77% reduction in

the level of lipoprotein in these cells. This was due to the resulting production of

thousands of copies of this modified msDNA molecule which apparently acts as

an antisense DNA in vivo to reduce the expression of the lpp gene. Other potential

uses of the versatile retron-msDNA system can be envisioned that have not been

explored experimentally. This might include a system for producing cDNA in

vivo, or over-production of a competing DNA binding site for a regulatory protein

(Lampson et al., 2001).

ACKNOWLEDGMENTS

I wish to thank Masayori Inouye, Sumiko Inouye, and Marlene Belfort for very

helpful comments and suggestions.

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CHAPTER 24

DICER: STRUCTURE, FUNCTION AND ROLE

IN RNA-DEPENDENT GENE-SILENCING PATHWAYS

JUSTIN M. PARE

AND TOM C. HOBMAN

12∗

Departments of Cell Biology and

Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada

∗

tom.hobman@ualberta.ca

1. INTRODUCTION

Dicer enzymes are double strand (ds)-specific RNases whose cleavage products

are used to provide specificity in RNA-based gene-silencing pathways. They are

encoded by the genomes of all multicellular and most unicellular eukaryotes. In

mammals, Dicer is essential for viability. Evidence suggests that these modular

200 kD enzymes function in the cytoplasm in conjunction with Dicer-binding

proteins to generate and load small RNAs onto ribonucleoprotein complexes that

effect post-transcriptional gene-silencing. Dicer activity is also required for some

types of transcriptional gene-silencing in the nucleus. The importance of Dicer as

a central regulator of gene expression is illustrated by recent estimates that the

expression of as many as one third of all human genes are regulated by Dicer

cleavage products (Lewis et al., 2005). In this chapter, we discuss the structure and

function of Dicer and related enzymes in RNA-based gene-silencing pathways.

2. STRUCTURE AND FUNCTION

2.1. RNase III Family Members

Dicer is a core member of the RNA interference (RNAi) apparatus that effects

gene-silencing at transcriptional and post-transcriptional levels. It belongs to a

large family of ribonucleases known as the RNase III superfamily. There are three

subfamilies within this superfamily, each represented by their type members: (1)

Escherichia coli RNase III, (2) Drosha and (3) Dicer. The subfamilies differ consid-

erably with respect to their structural complexity and functional roles within the cell.

421

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 421–438.