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Penghapusan Urutan Intron oleh Splicing RNA Sebagian besar, tetapi tidak semua, gen eukariota

yang lebih tinggi mengandung urutan intervensi moncoding atau intron sepa menilai urutan
pengkodean atau ekson (Lihat Bab 13 untuk diskusi tentang struktur halus gen eukariotik.) Lebih
sedikit, tetapi masih banyak dari gen eukariota bawah seperti ragi dan Neurospora mengandung
intron nonkode. Gen-gen langka dari beberapa virus prokariota, misalnya, bakteriofag E. coli T4 dan
B subtilis bakteriofag SPO1, dan dari archebacte- rium (bakteri primitif) juga telah ditemukan
mengandung intron. Dalam kasus gen "terbelah" atau "mosaik ini (urutan pengkodean dipisahkan
oleh urutan nonkode), transkrip primer berisi seluruh urutan gen dan urutan nonkode"
disambungkan "selama pemrosesan (Gbr. 10.23). ( Satuintron fag T4 tampaknya merupakan
pengecualian, bukti menunjukkan bahwa intron ini entah bagaimana dilewati selama proses
transkripsi.) Jelas, mekanisme penyambungan harus sangat canggih, ia harus menggabungkan
sekuens ekson dengan nukleotida tunggal akurat untuk memastikan bahwa kodon dalam ekson jauh
intron dibaca dalam bingkai bacaan yang benar. Akurasi pada tingkat ini tampaknya membutuhkan
sinyal splicing yang tepat, barangkali urutan nukleotida di dalam intron dan di persimpangan ekson-
intron. Namun, dalam transkrip primer gen nuklir hewan tingkat tinggi, satu-satunya sekuens yang
benar-benar dilestarikan adalah intron dinukleotida di ujung intron, yaitu, Urutan yang ditunjukkan
di sini adalah untuk untai DNA yang setara dengan transkrip RNA (T sebagai pengganti U) I Selain itu.
ada urutan konsensus pendek di persimpangan ekson-intron. Untuk gen nuklir, persimpangan konsis
adalah Subskrip numerik menunjukkan jumlah persentase basis konsensus di setiap posisi. dengan
demikian 100 subskrip menunjukkan bahwa basis selalu ada ai
Removal of Intron Sequences by RNA Splicing Most, but not all, genes of higher eukaryotes contain
moncoding intervening sequences or introns sepa rating the coding sequences or exons (See Chapter
13 for a discussion of the fine structure of eukaryotic genes.) Fewer, but still many, of the genes of
lower eukaryotes such as the yeasts and Neurospora contain noncoding introns. Rare genes of a few
viruses of prokaryotes, for example, E. coli bacteriophage T4 and B subtilis bacteriophage SPO1, and
of an archebacte- rium (a primitive bacterium) have also been found to contain introns. In the case
of these "split" or "mosaic genes (coding sequences separated by noncoding se quences), the
primary transcript contains the entire sequence of the gene and the noncoding sequences are
"spliced" out during processing (Fig. 10.23). (One phage T4 intron appears to be an exception,
evidence suggests that this intron is somehow bypassed during the transcription process.) Clearly,
the splicing mechanism must be very pre cise, it must join exon sequences with accuracrote single
nucleotide to assure that codons in exons dista to introns are read in the correct reading frame.
Accu- racy to this degree would seem to require ver precise splicing signals, presumably nucleotide
sequences within introns and at the exon-intron junctions. How ever, in primary transcripts of
nuclear genes of higher animals, the only completely conserved sequences different introns are the
dinucleotide sequences at the ends of introns, namely, The sequences shown here are for the DNA
strand that is equivalent to the RNA transcript (T in place of U)I addition. there are short consensus
sequences at the exon-intron junctions. For nuclear genes, the conse sus junctions are The numerical
subscripts indicate the percentugee quencies of the consensus bases at each position. thus a 100
subscript indicates that a base is always present ai

that position. N indicates that any of the four standard nucleotides may be present at the indicated
postson The exon-intron junctions are different in the case of RA genes and structural genes in
mitochordna and se olloring sectuon There is oni nuclear zeses ine so-called TACTAAC
box and it s rather poorly conserved The TACTAAC bok does exhibit a strong preference for either a
purine or a pyrimidine at each site as follows The adene resue a position six in the TACTAAC box
ismplerel sonserved and is known to plz ke len the splicing reacion (see the section Pre-mVA spiicing
RNAS, SRVPs, and the Splices some later in this chapter). The remaining sequences of introns foften
ver large) of nuclear genes are highly divergent and appear to be entirely random sequences. The
introns of genes of mitochondria and chloroplasts contain conserved sequences that are dif ferenm
iom those of nuclear genes Three Distinct T pes of RNA Splicing The scoei roncoding introns in genes
stimu- latec inserse mserest in the mechanism(s) by which intrun sequences ire removed during
gene expres sion. The early demonstration that the intron quences in eukarotic genes were
transcribed along

with the exon sequences focused research on the processing of primary gene transcripts. Just as in
vitro transcription and translation svstems were instrumen- n elucidating those processes, the key to
decipher- ing RNA splicing events was the development of in uttro splicing systems. As it turned out,
there are three totally distinct types of intron excision from RNA tran- scripts. We present them in
the order of increasing complexity, not in order of importance Class 3 introns are the most prevalent
and une most important in terms tal i of overall eukaryotic gene expression. I. The introns of tRNA
precursors are excised by precise endonucleolytic cleavage and ligation reactions cata lyzed by
special splicing endonuclease and ligase activ ities 2. The introns of Tetrahnymena RNA precursors
are re moved autocatalytically in a unique reaction mediated by the RNA molecule itself (no protein
enzymatic activ ity is involved) s. The introns of nuclear pre-mRNA (hnRNA) transcripts are spliced
out in rwo-step reactions carried out by complex ribonucleoprotein particles called spliceo- somes
RNA splicing mechanisms are the focus of consid erable research at present. and it is likely that nen
variations of these splicing mechanisms will be discov ered in the future Mans genes contain large
numbers of introns (eg. the chicken a2 collagen gene contans over 50 introns) which leads to the
question of the order in which multiple introns are removed. For certain genes that have been
studied, the introns are excised in a preferred, but not fixed, order. Other introns have been found
to undergo alternative path- ways of splicing leading to mRNAs that produce differ- ent proteins (i.e.,
with some common and some dis tinct amino acid sequences). Finally, one intron in the cytochrome
b gene of yeast mitochondria includes part of the coding sequence for a protein, an "RNA matu-
rase," that is responsible for excising the second intron from the transcript of that gene. This
suggests interest- ing mechanisms for regulating the expression of genes at the level of intron
processing. Clearly, then, inter esting variations in the use, structure, and excision of intron
sequences exist in nature, and novel intron structures are almost certain to be discovered in the
future. The three major established types of intron excision are described briefly in the following
three sections Splicing tRNA Precursors: Unique Nuclease and Ligase The tRNA precursor splicing
reaction has been worked out in detail in the yeast Saccharomyces cerevisiae. Both in vitro splicing
systems and temperature-sensi- tive splicing mutants have been used in dissecting the tRNA splicing
mechanism in S. cerer isiae. The excision of introns from veast tRNA precursors occurs in mo stages
(Fig. 102+) First, a nuclear membrane.bound splicing endonuclease makes tuo cuts precisely at the
ends of the intron Then. in a fairly complex set ot reactions, a splicing ligase joins tbe two balves of
the tRNA to produce the mature form of the tRNA mol- ecule The specificity for these reactions
resides in the conserved three-dimensional structural features of the tRNA precursors, not in the
nucleotide sequences per se Cleavage of the tRNA precursor yields 5'-OH ter mini and 2'-3' cyclic
phosphate groups at the 3' termini. The stage II ligation process actually involves four separate
reactions. (1) The first reaction is the addition of a phsophate group to the 5'-OH terminus this
reaction requires kinase activity and a phosphate donor (ATP). (2) Then, the 5' phosphate group is
activated by the transfer of an AMP group to the terminus from an AMP-ligase intermediate (the
originally having been derived from ATP also). (3) The 2'-3' cyclic phosphate is opened by a cyclic
phospho- diesterase activity that produces a 2' phosphate and a free 3' hydroxyl. (4) The final
ligation reaction occurs via a nucleophilic attack of the free 3'-OH on the interior 5' phosphate with
the release of AMP. All four of these reactions are catalyzed by the splicing ligase. Finally, the 2'
phosphate group (remaining from the 2'-3' cyclic phosphate produced by the original cleav- age
reaction) is removed by a phosphatase activity to yield the mature tRNA molecule. The overall two-
stage mode of tRNA intron exci- sion appears to occur in other organisms as wel!. In fact, the
mechanism mav involve the same reactions in plants. However, in mammais, the reactions are not
the same. Splicing still occurs in two stages, but the liga- tion reaction appears to directly join the 2'-
3 cyclic phosphate terminus to the 5'-OH terminus. The details of this process of tRNA precursor
splicing in mamma- lian cells are not yet as clearly established as are in yeast Autocatalytic Spilicing
of Tetrabhymena rRNA Precursor A general theme in biology is that metabolism occurs via
sequences of enzyme-catalyzed reactions. More- over, these all-important enzymes are generally
pro- teins, albeit sometimes single polypeptides and some- times complex heteromultimers, that
occasionally require nonprotein cofactors to perform their func- tions. Therefore, when covalent
bonds are being tered (removed, transferred, or formed), we expect that the reaction is being
catalyzed by an enzyme. Thus, the discovery that the intron in the rRNA precursor of Tetrahymena
thermophila was excised without the involvement of any protein was quite surprising to most
biologists. However, it is now clearly established that the splicing activity that excises the intron from
this rRNA precursor is intrinsic to the RNA molecule itself Moreover, such self-splicing or
autocatalytic activiby bas been shown to occur in rRNA precursors of several lower cularyotes and in
z large number o rRNA, IRNA, and mRNA precursors in and chloroplasts of many diferent species. In
the case of many of these introns (the so-called group I introns) in RNA precursor molecules, the sell
splicing mecha nism is the same as or very similar to that for Teraby mena rRNA precursors
described nexa For ochers (called group Il introns), the self splicing mechanism s similar to the
splicing mechanism observed with nuclear mRNA precursors (see the following section) excepx that
it requires no spliceosome The autocatalytic excision of the intron in the Tetrałymena rRNA
precursor (and other group I in- trons) requires no exzemal energy source (no ATP etc) and no
protein Instead, it involves a series of phospboester bond transfers with no bonds lost or gained in
the process The reaction requires a nine nucleoside or nucleotide with a free 3 OH group (GTP, GDP
GMP, or guanosine all work) as a cofactor plus a monovalent cation and a divalent a tion The
requirement for the G3' OH is absolute, no other base can be substituted in the nucleoside or
nucleotide cofactor Tbe intron is excised by means of tuo pbosphoester bond transfers and the
excised intron can subsequently circularize by means of an- other phosphoester bond transfer These
reactions are diagrammed in Fig 10.25 The autocatalytic circulariza tion of the excised intron
suggests that the self splicing of these rRNA precursors resides prirainly if not enturely, within the
intron structure itself Presumably the autocatalytic activity is dependent on the secondary structure
of the intron or at least the secondary struc ture of the RNA precursor molecule The secondary
structures of these self splicing RNAS must bring the groups into close juxaposition to allow the
phosphoester bond transfers to occur. Since the self splicing phosphoeser bond transfers are
potentiall reversible reacuons, rapid degradation of the excised introns or export of the spliced
rRNAs to the cyroplasm may drive splicing in the forward direction à key point is that the
autocatalytic splicing reactions are intramolecular in nature and, thus, are not concentration
dependent Moreorer, the RNA pre cursors are capable of forming an actite center in which the
guanosine 3 OH cofactor binds Thus catalytic sites are not restricted to proteins: but also note that
there is no rans catal tic activity as for enzvmes, only cis catalytic activity Pre-mRNA Splicing: snRNAs,
snRNPs. and the Spliceosome The introns in nuclear mRNA precursors (nuclear pre-mRNAs) are
excised in two steps like the introns in yeast pre-tRNAs and Tetrahymena pre-rRNAs discussed

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