Indian Journal of Biochemistry & Biophysics Vol. 38, October 2001 , pp.289-293

Mini Review

A unique group of self-splicing introns in bacteriophage T4

Asad U Khan *l, Malik Ajama\uddin' and Masood Ahmad#

I Interdisciplinary Biotechnology Unit. A M U Aligarh 202 002, Indi a

# Biochemistry Department Faculty of Life Sciences, A M U Aligarh 202 002, India

Received 1 February 2001: accepted 18 May 2001

We describe in this review, the salient splicing features of group I introns of bac teriophage T4 and propose, a hypothetical model to fit in the self-splic ing of nrdB intron of T4 phage. Occurrence of non-coding sequences in prokaryotic cell s is a rare event while it is common in ellkaryotic cells, especially the higher eukaryotes. Therefore. T4 bacteriophage can serve as a good model system to study the evoluti onary aspects of splicing of introns. Three genes of T4 phage were fou nd to have st retches of non-coding sequences which belonged to the group IA type introns of self-splici ng nature.

Discovery of introns in 1977 heralded a new era in the study of molecular biology of eukaryotic gene expression I. The complexity of gene organization, the combinatorial possibilities of assembling different coding exons from an RNA precursor, and the novelty of the RNA splicing process clearly indicate that eukaryotic molecular biology would be fasc inatingly different from that of prokaryotic system2

.3 . But Mon'issey and Tollervey have highlighted a striking similarity between prokaryotic and eukaryotic systems4 th at ribosomal RNA processing in eukaryotes, archaebacteria and bacteria is of common origin can be shown by the fact that the pre-rRNA processing is fo und in archaeon, Solfolobus acidocaldarius5

.6

.

In general, prokaryotic genes do not have introns because of energetic demands of rapid generation time, coupled with single origin of replication7

. Therefore, it is thought that introns and other non-coding DNA sequences have been kept out of prokaryotes. But an exception to this is the occurrence of three introns in bacteriophage T4. Group I introns have been found in mitochondrial and chloroplast genomes of plants, fungi, a few nuclear rRNA, bacteriophages and also in some fungal plasmids8

. Based on all known group I intron sequences, comparative folding analyses and computer search for structure with minimum free energy of folding were performed and generic

d d . d . d9 10 secon aryan tertiary structures were enve . .

* Author for correspondence at hi s present address: Biochemi stry Department, RWJMS , University of Medicine and Dentistry New Jersey 675 Hoes Lane, Pi scataway, New Jersey 08854-5635. USA; E mail: asaduk@lIsa.net

T4 introns The bacteriophage T4 genome contains three

introns (Fig. I) , all of which reside in genes involved in nucleotide biosynthesi s. The first T4 intron was discovered in 1984 in the tel gene coding for thymidylate synthase t I , whereas the other two introns are in the nrdB gene coding for small subunit of ribonucleotide reductase l2 and the nrelD gene coding for small subunit of anaerobic ribonucleoside triphosphate reductase l3

. The reason of the occurrence of introns only in the genes involved in nucleotide biosynthesis is still unanswered. These introns differ in sequence and size; the nrelD and tel introns comprise 1033 and 1016 nucleotides respectively, whi le IlrdB intron has only 598 nucleotides. All these introns comprise Open Reading Frames (ORFs). The ORFs are not homologous and occur at three different positions, at least two of which looped out from a secondary structure. Autocatalysis of these introns is enabled by the folding of introns into characteristic secondary structures required for intron excision and exon ligation 10. 14. The critical ribozyme secondary structure comprises 10 RNA pairing regions (PI-PIO) which alongwith conserved primary sequence elements namely P, Q, Rand S identify the T4 .. I . 15 t 6 Th P4 1I1tervemng sequences as group 1I1tron · . e .. P6 domain directs the folding of Tetrahymena ribozyme core 17. It is not established that whether the tertiary structure of group I intron is essential for catalytic activity. Basic features of predicted three dimensional (3 D) tertiary structure have been studied by different techniques like photochemical cross­linking reactions, affinity cleavage reagents, NMR

d I · h 18·22 spectroscopy an computer a gont m .

290

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INDIAN J BIOCHEM. BIOPHYS., VOL. 38, OCTOBER 2001

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Fi g. I-Secondary structure for intron sequences from the IIrdB , Id and SUllY (nrclD) genes

KHAN el aJ.: A UNIQUE GROUP OF SEL-SPLICING INTRONS IN BACTERIOPHAGE T4 291

Self splicing of group I intron

Cech and coworkers23 found that a ribosomal precursor RNA of Tetrahymena could remove its own 413 nucleotides long intron in the absence of proteins in vitro. This reaction proceeds by two consecutive trans-esteri fication reactions and requires a di valent

t· (M 2+ M 2+) . ca IOn g or n as well as guanosIne (or a guanosine phosphate)24. Divalent cation is needed for the formation of stable secondary structure of group I . t 25.26 R I' h b f In ron . ecent y It as een ound that Cobalt ion [Co(III)(NH3)6]3+ (ref. 27-29) and other monovalent cation (Lt or even NH/) at higher concentration can

b · h M 2+ su stltute t e g (ref. 30). These findings reveal that the fundamental requirement for RNA catalysis is simply one of a relatively positive charge. The 3' end of the 5' exon forms a hybrid with an internal guide sequence (lGS) in the 5' end of the intron. The trans­esterification reaction is initiated by the external guanosine which is held in a guanosine binding site of the intron hydrogen bridges31. The 3'-OH of this g.uanosine attacks the phosphorus atom at the 5' splice site and forms 3',5'-phosphodiester bond with the first nucleotide of intron. In the second trans-esterification step, the free 3' hydroxyl group at the 3' splice site brings about the ligation of exons and removal of the linear intron (linear intervening sequences), while the extra guanosine remains at its 5' end. During this reaction, the conserved 3' guanosine from the intron remains bound at the G-binding site. Subsequently, the intron itself undergoes a self-catalyzed trans­esterification reaction. The conserved terminal 3' guanosine residue still bound in the G-binding site attacks a phosphorus atom near the end of the molecule resulting in intron cyclization (cyclic intervening sequences) and removal of short 5' 01 igonucleotide32.

Self-splicing of group I intron is very sensitive to mu~ation in core of this structure but many of the penpheral stem-loop structures can be deleted without loss of splicing function in vivo. These structures may, however, play a role in stabilizing the intron or providing binding sites for proteins which facilitate or ~egulate self splicing in vivo. Introns themselves may In fact encode a protein, involved in their own structural stabi lization33.

Splicing of T4 introns The T4 introns have the ability to self-splice in

't 13.34 A I" b Vl ro . utocata YSls IS ena led by folding of the intron into characteristic secondary structure that facilitates a series of trans-esterification reactions

necessary for intron excision and ex on ligation8. The T4 td intron contains four long range base paired regions in P3 , P6, P7 and PI 0 . The remaining base paired regions are stem-loop structure including P7.1 and P7.2 which coupled with specific variants in the P, Q, Rand S sequence elements further sub classify the T4 introns as group IA 8.15 .

The secondary structure model for group I intron was initially postulated on the basis of phylogenetic comparisons of intron sequences from various organisms which predicted a common secondary structure35.36. Since then, mutational analysis of autocatalytic group I intron in Tetrahymena, T4 and yeast mitochondria has identified many splicing defective cis-acting intron mutations that would destabilize p,redicted RNA pairing regions, thus generally supporting the model. In T4, for example, the sequence changes for 31 splicing defecti ve td intron mutants (19 phage mutants, 12 mutations from a cloned td gene) have been determined and the vast majority disrupt proposed intron RNA helices 37.39.

Single base changes in predicted RNA pairing regions which disrupt splicing, provides proof that a nucleotide is essential for the splicing reaction40.41. Genetic verification that a base pair (and thus a pairing region) exists, can be demonstrated by the isolation of a pseudorevertant that harbours a compen­satory mutation at the nucleotide predicted to base pair with the original mutant nucleotide. The double mutant would be predicted to regenerate a Watson­Crick base pair in an essential RNA helix, thus permitting intron RNA to fold, resulting in the restoration of some degree of autocatalytic splicing42. Such experiments have been carried out using si te­~pecific mutagenesis to verify the existence of group I Intron RNA pairing region in Tetrahymena and yeast

' t h d ' 43-45 F . ml oc on na . or the T4 Introns, however, only the P6 pairing region of the td intron has been proven by isolation of such second site suppressor mutations38.

Conclusion Our study demonstrated that 3' region of ORF in

the nrdB intron could also play significant role in I·· 40 sp ICIng . In contrast, genetic studies on td and other

group I introns demonstrated that intron ORF is not required for active splicing conformation39. This could probably be due to the small size of ORF of nrdB intron (294 bases) compared to that of td intron (778 bases) which looped out of the secondary structure models for RNA folding26

,46 . Present study of the nrdB mutants of phage T4 and their revertants

292 INDIAN J BlOCH EM. BIOPHYS., VOL. 38, OCTOBER 200 1

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Fig. 2-A proposed model for tertiary structure of nrdB intron involved in self-splicing. [Solid lines show connectivities with each RNA domain whi le dashed lines indicate con nect ivities within the intact intron. Arrows superimposed on lines give 5 ' to 3' polarity . The 5' and 3' exon sequences are shown in lower case le tters and both sites are marked with arrows. Base-paired segments are designated as "P" and sec tion join the two paired region as "1")

further leads us to conclude that the conserved sequences of nrdB intron that are important in pre­mRNA spli cing, especially the extreme ends of nrdB intron, were more critical for auto-catalysis , Moreover, the exon sequences immediately adjacent to the sp lice site were also important for splice site se lection40

.42. Finally, the data also strongly suggest that the cleavage of intron might require a well defined secondary structure which on folding would probably acquire a typical tertiary structure involving the P9.0 and PI 0 loops. These secondary and lor tertiary structures are crucial for the splicing of nrdB intron like other group I type of introns47

.

In view of the above discussion, we would like to propose a pLausible model to show the in volvement of various domains of nrdB intron resulting in the formation of typical tertiary structure (Fig. 2 ). This hypothetical model for the auto-catalysis of nrdB

intron is very close to that proposed earlier in case of 'T' I . 48 1 elra lymena \Otron . References

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