Lesson 4

Transcription in Eukaryotic cells

• We have learned that bacteria have only one RNA polymerase, wh
ich makes all three of the familiar RNA types: mRNA, rRNA, and
tRNA. True, the polymerase can switch σ-factors to meet the dem
ands of a changing environment, but the core enzyme remains ess
entially the same. Quite a different situation prevails in the eukary
otes. In this lesson we will see that three distinct RNA polymerase
s occur in the nuclei of eukaryotic cells. Each of these is responsi
ble for transcribing a separate set of genes, and each recognizes a
different kind of promoter.
Multiple Forms of Eukaryotic
RNA Polymerase
• Several early studies suggested that at least two RNA polymerases operate in
eukaryotic nuclei: one to transcribe the major ribosomal RNA genes (those c
oding for the 28S, 18S, and 5.8S rRNAs in vertebrates), and one or more to tr
anscribe the rest of the nuclear genes.
• To begin with, the ribosomal genes are different in several ways from other n
uclear genes: (1) They have a different base composition from that of other n
uclear genes. For example, rat rRNA genes have a GC content of 60%, but th
e rest of the DNA has a GC content of only 40%.
• (2) They are unusually repetitive; depending on the organism, each cell conta
ins from several hundred to over 20,000 copies of the rRNA gene. (3) They a
re found in a different compartment—the nucleolus—than the rest of the nucl
ear genes. These and other considerations suggested that at least two RNA po
lymerases were operating in eukaryotic nuclei. One of these synthesized rRN
A in the nucleolus, and the other synthesized other RNA in the nucleoplasm
(the part of the nucleus outside the nucleolus).
 Separation of the Three
Nuclear Polymerases
• Robert Roeder and William Rutter showed in 1969 that eukary
otes have not two, but three different RNA polymerases. Furthe
rmore, these three enzymes have distinct roles in the cell. Thes
e workers separated the three enzymes by DEAE-Sephadex io
n-exchange chromatography. They named the three peaks of po
lymerase activity in order of their emergence from the ion-exc
hange column: RNA polymerase I, RNA polymerase II, and
RNA polymerase III
• The three enzymes have differ
ent properties besides their dif
ferent behaviors on DEAESep
hadex chromatography. For ex
ample, they have different res
ponses to ionic strength and di
valent metals. More important
ly, they have distinct roles in tr
anscription: Each makes differ
ent kinds of RNA.
• Roeder and Rutter next looked in
purified nucleoli and nucleoplasm
to see if these subnuclear compart
ments were enriched in the appro
priate polymerases. polymerase I
is indeed located primarily in the
nucleolus, and polymerases II and
III are found in the nucleoplasm.
This made it very likely that poly
merase I is the rRNA-synthesizin
g enzyme, and that polymerases II
and III make some other kinds of
RNA.
The Roles of the Three R
NA Polymerases
• How do we know that the three RNA polymerases have diff
erent roles in transcription? The clearest evidence for these
roles has come from studies in which the purifi ed polymer
ases were shown to transcribe certain genes, but not others,
in vitro. Such studies have demonstrated that the three RN
A polymerases have the following specificities. Polymeras
e I makes the large rRNA precursor. In mammals, this prec
ursor has a sedimentation coeffi cient of 45S and is process
ed to the 28S, 18S, and 5.8S mature rRNAs. Polymerase II
makes an ill-defi ned class of RNA known as heterogeneou
s nuclear RNA (hnRNA) as well as the precursors of micro
RNAs (miRNAs) and most small nuclear RNAs (snRNAs).
• Most of the hnRNAs are precursors of mRNAs and that the snR
NAs participate in the maturation of hnRNAs to mRNAs. micro
RNAs control the expression of many genes by causing degrada
tion of, or limiting the translation of, their mRNAs. Polymerase
III makes precursors to the tRNAs, 5S rRNA, and some other s
mall RNAs
 RNA Polymerase Subunit
Structures
• The first subunit structures for a eukaryotic RNA polymerase (poly
merase II) were reported independently by Pierre Chambon and Rut
ter and their colleagues in 1971, but they were incomplete. We shou
ld note in passing that Chambon named his three polymerases A, B,
and C, instead of I, II, and III, respectively. However, the I, II, III no
menclature of Roeder and Rutter has become the standard. We now
have very good structural information on all three polymerases from
a variety of eukaryotes. The structures of all three polymerases are q
uite complex, with 14, 12, and 17 subunits in polymerases I, II, and
III, respectively. Polymerase II is by far the best studied, and we wil
l focus the rest of our discussion on the structure and function of tha
t enzyme.
 Polymerase II Structure
• For enzymes as complex as the eukaryotic RNA polymeras
es it is difficult to tell which polypeptides that copurify wit
h the polymerase activity are really subunits of the enzyme
s and which are merely contaminants that bind tightly to th
e enzymes. One way of dealing with this problem would b
e to separate the putative subunits of a polymerase and the
n see which polypeptides are really required to reconstitute
polymerase activity. Although this strategy worked beautif
ully for the prokaryotic polymerases, no one has yet been a
ble to reconstitute a eukaryotic nuclear polymerase from it
s separate subunits. Thus, one must try a different tack.
• Another way of approaching this problem is to find the genes for all
the putative subunits of a polymerase, mutate them, and determine
which are required for activity. This has been accomplished for one
enzyme: polymerase II of baker’s yeast, Saccharomyces cerevisiae.
Several investigators used traditional methods to purify yeast polym
erase II to homogeneity and identified 10 putative subunits. Later, s
ome of the same scientists discovered two other subunits that had be
en hidden in the earlier analyses, so the current concept of the struct
ure of yeast polymerase II includes 12 subunits. The genes for all 12
subunits have been sequenced, which tells us the amino acid sequen
ces of their products. The genes have also been systematically mutat
ed, and the effects of these mutations on polymerase II activity have
been observed.
• Human and Yeast RNA Polymerase II Subunits
• Each of these polypeptides is encoded in a single gene in the yea
st and human genomes. The names of these polymerase subunits,
Rpb1, and so on, derive from the names of the genes that encode
them (RPB1, and so on). Note the echo of the Chambon nomencl
ature in the name RPB, which stands for RNA polymerase B (or
II).
• How do the structures of polymerases I and III compare with this poly
merase II structure? First, all the polymerase structures are complex—
even more so than the structures of the bacterial polymerases. Second,
all the structures are similar in that each contains two large (greater th
an 100 kD) subunits, plus a variety of smaller subunits. In this respect,
these structures resemble those of the prokaryotic core polymerases,
which contain two high-molecular-mass subunits (β and β’) plus three
low-molecular-mass subunits
• (two α’s and an ω). In fact, as we will see later in this chapter, an evol
utionary relationship is evident between three of the prokaryotic core
polymerase subunits and three of the subunits of all of the eukaryotic
polymerases. In other words, the three eukaryotic polymerases are rel
ated to the prokaryotic polymerase and to one another.
• the three yeast nuclear polymerases have several subunits i
n common. In fact, five such common subunits exist. In th
e polymerase II structure, these are called Rpb5, Rpb6, Rp
b8, Rpb10, and Rpb12.
 Core Subunits

• These three polypeptides, Rpb1, Rpb2, and Rpb3, are all a

bsolutely required for enzyme activity. They are homologo
us to the β’-, β-, and α-subunits, respectively,
• of E. coli RNA polymerase.
• How about functional relationships? We have seen that the
E. coli β’-subunit binds DNA, and so does Rpb1. The E. co
li β-subunit is at or near the nucleotide-joining active site o
f the enzyme. Using the same experimental design, André
Sentenac and his colleagues have established that Rpb2 is
also at or near the active site of RNA polymerase II. The fu
nctional similarity among the second largest subunits in all
three nuclear RNA polymerases, as well as prokaryotic pol
ymerases, is mirrored by structural similarities among thes
e same subunits, as revealed by the sequences of their gene
s.
• Although Rpb3 does not closely resemble the E. coli a-sub
unit, there is one 20-amino-acid region of great similarity.
In addition, the two subunits are about the same size and h
ave the same stoichiometry, two monomers per holoenzym
e. Furthermore, the same kinds of polymerase assembly de
fects are seen in RPB3 mutants as in E. coli a-subunit muta
nts. All of these factors suggest that Rpb3 and E. coli a are
homologous.
• Common Subunits Five subunits—Rpb5, Rpb6, Rpb8, Rpb
10, and Rpb12—are found in all three yeast nuclear polym
erases. We know little about the functions of these subunits
, but the fact that they are found in all three polymerases su
ggests that they play roles fundamental to the transcription
process.
 Promoters

• We have seen that the three eukaryotic RNA polymerases h

ave different structures and they transcribe different classe
s of genes. We would therefore expect that the three polym
erases would recognize different promoters, and this expec
tation has been borne out. We will conclude this chapter by
looking at the structures of the promoters recognized by all
three polymerases.
 Class II Promoters
• We begin with the promoters recognized by RNA polymerase II (cla
ss II promoters) because these are the most complex and best studie
d. Class II promoters can be considered as having two parts: the core
promoter and the proximal promoter. The core promoter attracts gen
eral transcription factors and RNA polymerase II at a basal level and
sets the transcription start site and direction of transcription. It consi
sts of elements lying within about 37 bp of the transcription start site
, on either side. The proximal promoter helps attract general transcri
ption factors and RNA polymerase and includes promoter elements t
hat can extend from about 37 bp up to 250 bp upstream of the transc
ription start site. Elements of the proximal promoter are also someti
mes called upstream promoter elements.
• The core promoter is modular and can contain almost any combinatio
n of the following elements . The TATA box is centered at approximat
ely position 228 (about 231 to 226) and has the consensus sequence T
ATA(A/T)AA(G/A); the TFIIB recognition element (BRE) lies just up
stream of the TATA box (about position 237 to 232) and has the conse
nsus sequence (G/C)(G/C)(G/A)CGCC; the initiator (Inr) is centered
on the transcription start site (position 22 to 14) and has the consensus
sequence GCA(G/T)T(T/C) in Drosophila, or PyPyAN(T/A)PyPy in
mammals; the downstream promoter element (DPE) is centered on
position 130 (128 to 132); the downstream core element (DCE) has t
hree parts located at approximately 16 to 112, 117 to 123, and 131 to
133, and these have the consensus sequences CTTC, CTGT, and AGC
, respectively; and the motif ten element (MTE) lies approximately b
etween positions 118 and 127.
• A generic class II core promoter. This core promoter contai
ns up to six elements. These are, 59 to 39: the TFIIB-recog
nition element (BRE, purple); the TATA box (red); the initi
ator (green); the downstream core element, in three parts
(DCE, yellow); the motif ten element (MTE, blue); and the
downstream promoter element (DPE, orange). The exact lo
cations of these promoter elements are given in the text.
 Class I Promoters

• What about the promoter recognized by RNA polymerase I? We can r

efer to this promoter in the singular because almost all species have o
nly one kind of gene recognized by polymerase I: the rRNA precursor
gene. The one known exception is the trypanosome, in which polymer
ase I transcribes two protein-encoding genes, in addition to the rRNA
precursor gene. It is true that the rRNA precursor gene is present in hu
ndreds of copies in each cell, but each copy is virtually the same as th
e others, and they all have the same promoter sequence. However, this
sequence is quite variable from one species to another—more variable
than those of the promoters recognized by polymerase II, which tend t
o have conserved elements, such as TATA boxes, in common.
 Class III Promoters

• As we have seen, RNA polymerase III transcribes a variety of

genes that encode small RNAs. These include (1) the “classica
l” class III genes, including the 5S rRNA and tRNA genes, and
the adenovirus VA RNA genes; and (2) some relatively recently
discovered class III genes, including the U6 snRNA gene, the 7
SL RNA gene, the 7SK RNA gene, and the Epstein–Barr virus
EBER2 gene. The latter, “nonclassical” class III genes have pr
omoters that resemble those found in class II genes. By contras
t, the “classical” class III genes have promoters located entirely
within the genes themselves.
Class III Genes with Internal Promote
rs
• Donald Brown and his colleagues performed the fi rst anal
ysis of a class III promoter, on the gene for the Xenopus bo
realis 5S rRNA. The results they obtained were astonishing
. Whereas the promoters recognized by polymerases I and
II, as well as by bacterial polymerases, are located mostly i
n the 5’-flanking region of the gene, the 5S rRNA promote
r is located within the gene it controls.
• Promoters of some class III genes. The promoters of the 5S
, tRNA and U6 RNA genes are depicted as groups of blue
boxes within the genes they control. DSE and PSE are dist
al and proximal sequence elements, respectively
 Class III Genes with Class II-like Pro
moters
• After Brown and other investigators established the novel idea of
internal promoters for class III genes, it was generally assumed t
hat all class III genes worked this way. However, by the mid-198
0s some exceptions were discovered. The 7SL RNA is part of the
signal recognition particle that recognizes a signal sequence in ce
rtain mRNAs and targets their translation to membranes such as t
he endoplasmic reticulum. In 1985, Elisabetta Ullu and Alan Wei
ner conducted in vitro transcription studies on wild-type and mut
ant 7SL RNA genes that showed that the 59-flanking region was
required for high-level transcription. Without this DNA region, tr
anscription effi ciency dropped by 50–100-fold.
 Enhancers and Silencers

• Many eukaryotic genes, especially class II genes, are associated wi

th cis-acting DNA elements that are not strictly part of the promote
r, yet strongly infl uence transcription. Enhancers are elements that
stimulate transcription. Silencers, by contrast, depress transcriptio
n.

• Structure of the SV40 virus early control region. As usual, an arro

w with a right-angle bend denotes the transcription initiation site, a
lthough this is actually a cluster of three sites. Upstream of the star
t site we have, in right-to-left order, the TATA box (red), six GC bo
xes (yellow), and the enhancer (72-bp repeats, blue
General Transcription
Factors in Eukaryotes
• Eukaryotic RNA polymerases, unlike their bacterial counterparts, are
incapable of binding by themselves to their respective promoters. Inst
ead, they rely on proteins called transcription factors to show them th
e way. Such factors are grouped into two classes: general transcriptio
n factors and gene-specific transcription factors (activators). Without
activators, the general transcription factors can attract the RNA poly
merases to their respective promoters, but only to a weak extent. Ther
efore, these factors can support only a basal level of transcription. Fu
rthermore, general transcription factors and the three polymerases alo
ne allow for only minimal transcription control, whereas activators h
elp cells exert exquisitely fine control over transcription. Nevertheles
s, the task performed by the general transcription factors—getting the
RNA polymerases together with their promoters—is not only vital, b
ut also very complex because many polypeptides are required to do t
he job.
 Class II Factors
• Transcription factors bind to class II promoters, including the adenovirus
major late promoter, in the following order in vitro: (1) TFIID, apparentl
y with help from TFIIA, binds to the TATA box, forming the DA comple
x. (2) TFIIB binds next. (3) TFIIF helps RNA polymerase bind to a regio
n extending from at least position 234 to position 117. The remaining fact
ors bind in this order: TFIIE and TFIIH, forming the DABPolFEH preinit
iation complex. The participation of TFIIA seems to be optional in vitro.
TFIID contains a 38-kD TATA-box binding protein (TBP) plus several ot
her polypeptides known as TBP-associated factors (TAFs). The C-termin
al 180 amino acid fragment of the human TBP is the TATA-box-binding
domain. The interaction between a TBP and a TATA box takes place in th
e DNA minor groove. The saddle-shaped TBP lines up with the DNA, an
d the underside of the saddle forces open the minor groove and bends the
TATA box through an 80-degree curve angle.
• Model for the interaction between TBP and TATA containing or TATA-less promoters. (a) TATA-containing
promoter. TBP can bind by itself to the TATA box of this promoter (top). It can also bind in the company of
all the TAFs in TFIID (middle). And it can bind with a subset of TAFs (bottom). (b) TATA-less promoter wi
th initiator element and DPE. TBP cannot bind by itself to this promoter, which contains no TATA box (top)
. The whole TFIID is competent to bind to the TATA-less promoter through interactions between TAF1 (yel
low) and TAF2 (brown, middle). TAF1 and TAF2 are sufficient to tether TBP to the initiator and DPE (bott
om). (c) TATA-less promoter with GC boxes. TBP cannot bind to this promoter by itself (top). The whole T
FIID can bind to this promoter through interactions with Sp1 bound at the GC boxes (middle). TAF1, TAF2
, and TAF4 are sufficient to anchor TBP to the Sp1 bound to the GC boxes.
 Class I Factors
• Class I promoters are recognized by two transcription factors, a co
re-binding factor and a UPE-binding factor. The human core-bindi
ng factor is called SL1; in some other organisms, such as A. castell
anii, the homologous factor is known as TIF-IB. The core-binding
factor is the fundamental transcription factor required to recruit R
NA polymerase I. This factor also determines species specificity, a
t least in animals. The factor that binds the UPE is called UBF in
mammals and most other organisms, but UAF in yeast. It is an ass
embly factor that helps the core-binding factor bind to the core pro
moter element. The degree of reliance on the UPE binding factor v
aries considerably from one organism to another. In A. castellanii,
TIF-IB alone suffices to recruit the RNA polymerase I and positio
n it correctly for initiation of transcription.
 Class III Factors

• Transcription of all classical class III genes requires TFIII

B and C, and transcription of the 5S rRNA genes requires t
hese two plus TFIIIA.
Transcription Activators
in Eukaryotes
• we learned about the basic machinery involved in eukaryotic transcri
ption: the three RNA polymerases, their promoters, and the general t
ranscription factors that bring RNA polymerase and promoter togeth
er. However, it is clear that this is not the whole story. The general tr
anscription factors by themselves dictate the starting point and direct
ion of transcription, but they are capable of sponsoring only a very l
ow level of transcription (basal level transcription). But transcription
of active genes in cells rises above (frequently far above) the basal l
evel. To provide the needed extra boost in transcription, eukaryotic c
ells have additional, gene- specific transcription factors (activators) t
hat bind to DNA elements called enhancers. The transcription activa
tion provided by these activators also permits cells to control the exp
ression of their genes.
 Self study

• Chromatin Structure and Its Effects on Transcription

RNA Processing : Splicing, Capp
ing and Polyadenylation
• Bacterial gene expression can be summarized very briefly as follows:
First, RNA polymerase transcribes a gene, or set of genes, in an opero
n. Then, even while transcription is still occurring, ribosomes bind to t
he mRNA and translate it to make protein. We have already studied th
e transcription part of this scheme, and it may seem to be quite compl
ex. However, the situation in eukaryotes is much more intricate. In eu
karyotes, the compartments in which transcription and translation occ
ur are different. Transcription takes place in the nucleus, whereas trans
lation takes place in the cytoplasm. This means that transcription and t
ranslation cannot occur simultaneously as they do in bacteria. Instead,
transcription has to finish, then the transcript has to make its way into
the cytoplasm before translation can begin. This allows an interval bet
ween transcription and translation traditionally known as the posttrans
criptional phase.
• In this part we will see that most eukaryotic genes, in contrast to typic
al bacterial genes, are interrupted by noncoding DNA. RNA polymera
se cannot distinguish the coding region of the gene from the noncodin
g regions, so it transcribes everything. Thus, the cell must remove the
noncoding RNA from the original transcript, in a process called splici
ng. Eukaryotes also tack special structures onto the 5’- and 3’-ends of
their mRNAs. The 5’-structure is called a cap, and the 3’-structure is
a string of AMPs called poly(A). All three of these events occur in the
nucleus before the mRNA emigrates to the cytoplasm, and it is becom
ing increasingly clear that all three occur before transcription is over.
Thus, it might be more correct to refer to them as cotranscripional, rat
her than posttranscriptional, events. To avoid any confusion, we will r
efer to them as mRNA processing events. It appears that all three of th
ese events are coordinated.
Genes in Pieces

• Most higher eukaryotic genes coding for mRNA and tRNA

, and a few coding for rRNA, are interrupted by unrelated r
egions called introns. The other parts of the gene, surround
ing the introns, are called exons; the exons contain the seq
uences that finally appear in the mature RNA product. Gen
es for mRNAs have been found with anywhere from zero t
o 362 introns. Transfer RNA genes have either zero or one.
 RNA Splicing
• Consider the problem introns pose. They are present in genes but not
in mature RNA. How is it that the information in introns does not fi n
d its way into the mature RNA products of the genes? The two main
possibilities are: (1) The introns are never transcribed; the polymeras
e somehow jumps from one exon to the next and ignores the introns i
n between. (2) The introns are transcribed, yielding a primary transcri
pt, an overlarge gene product that is cut down to size by removing th
e introns. As wasteful as it seems, the latter possibility is the correct o
ne. The process of cutting introns out of immature RNAs and stitchin
g together the exons to form the final product is called RNA splicing.
The splicing process is outlined in the following Figure, although, as
we will see later in the chapter, this picture is considerably oversimpl
ified.
• Outline of splicing. The introns in a gene are transcribed along
with the exons (colored boxes) in the primary transcript. Then
they are removed as the exons are spliced together.
• Messenger RNA synthesis in eukaryotes occurs in stages.
The fi rst stage is synthesis of the primary transcription pro
duct, an mRNA precursor that still contains introns copied
from the gene, if any were present. This precursor is part o
f a pool of large nuclear RNAs called hnRNAs. The secon
d stage is mRNA maturation. Part of the maturation of an
mRNA precursor is the removal of its introns in a process
called splicing. This yields the mature sized mRNA.
 Splicing Signals
• Consider the importance of accurate splicing. If too little RNA is rem
oved from an mRNA precursor, the mature RNA will be interrupted b
y nonsense regions. If too much is removed, important sequences may
be left out.
• Given the importance of accurate splicing, signals must occur in the
mRNA precursor that tell the splicing machinery exactly where to “cu
t and paste.” What are these signals? One way to fi nd out is to look at
the base sequences of a number of different genes, locate the intron bo
undaries, and see what sequences are common to all of them. In princi
ple, these common sequences could be part of the signal for splicing.
The most striking observation, first made by Chambon, is that almost
all introns in nuclear mRNA precursors begin and end the same way:

• exon/GU–intron–AG/exon
• In other words, the first two bases in the intron of a transcript are GU
and the last two are AG. This kind of conservation does not occur by
accident; surely the GU–AG motif is part of the signal that says, “Spl
ice here.” However, a typical intron will contain several GU’s and A
G’s within it. Why are these not used as splice sites? The answer is th
at splicing signals are more complex than that. They contain sequenc
es at the exon-intron boundaries that extend beyond simply GU and
AG, and they include a “branchpoint” sequence near the 3’-end of th
e intron, which we will discuss later in this chapter. Sequencing of m
any genes has revealed the following mammalian consensus sequenc
es:
• 5’-AG/GUAAGU–intron–YNCURAC–YnNYAG/G-3’
• where the slashes denote the exon–intron borders, Y is either pyrimidine (U or C),
Yn denotes a string of about nine pyrimidines, R is either purine (A or G), A is a sp
ecial A in the “branchpoint” sequence within the intron, and N is any base.
• The splicing signals in nuclear mRNA precursors are rema
rkably uniform. The fi rst two bases of the intron are almos
t always GU, and the last two are almost always AG. The
5’- and 3’-splice sites have consensus sequences that exten
d beyond the GU and AG motifs, and there is also a branch
point consensus sequence. All three consensus sequences a
re important to proper splicing; when they are mutated, ab
normal splicing can occur
 Effect of Splicing on Gene Expression

• It seems obvious that splicing introduces a degree of ineffi

ciency into the gene expression process. Introns must be tr
anscribed, only to be immediately removed from pre mRN
Asand degraded. Moreover, inaccurate splicing can disrup
t an mRNA and lead to mistranslation. So it is fair to ask w
hy evolution has not eliminated splicing from eukaryotes. I
ndeed, introns are relatively rare and small in simple eukar
yotes like yeasts, but they are abundant and long— typicall
y much longer than exons—in higher eukaryotes, includin
g humans.
• One reason that splicing may have evolved to become so p
rominent in higher eukaryotes is that it actually facilitates
gene expression. In 2003, Shihua Lu and Bryan Cullen sur
veyed 10 human genes with and without introns in their 5’-
untranslated regions and found that the introns improved g
ene expression in every case—from a relatively modest tw
o-fold to about 3’-fold in the case of the b-globin gene, wh
ich actually depends on introns for efficient expression. Th
e advantage of introns comes from at least two sources: Th
ey stimulate efficient mRNA 3’-end formation, and they m
ake translation more efficient.
• It seems paradoxical that the presence or absence of introns could affect
translation, as translation occurs in the cytoplasm, long after the introns
have been removed. But we need to consider the fact that mRNAs do no
t exist as naked RNAs. Rather, they are complexed with a wide variety o
f proteins in the nucleus, and many of these proteins travel with the mR
NA as a messenger ribonucleoprotein (mRNP) as it is transported to the
cytoplasm. And some of the proteins are added to the mRNP at the exon
junctions during splicing to form the exon junction complex (EJC). Th
e presence of EJCs is necessary and sufficient for stimulation of gene ex
pression by introns, probably by facilitating the association of mRNAs
with ribosomes. Thus, it is the proteins added to the mRNP during splici
ng, rather than splicing itself, that causes the stimulation. In Chapter 18,
we will see that the EJC also makes possible the destruction of faulty m
RNAs that have premature stop codons. This also enhances efficiency b
y removing damaged mRNAs that would occupy ribosomes unproductiv
ely.
 Spliceosomes
• Edward Brody and John Abelson discovered in 1985 that the lariat-sh
aped splicing intermediates in yeast are not free in solution, but boun
d to 40S particles they called spliceosomes. These workers added lab
eled pre-mRNAs to cell-free extracts and used a glycerol gradient ult
racentrifugation procedure to purify the spliceosomes. Analysis of th
ese RNAs by electrophoresis revealed the presence of lariats: the spli
cing intermediate and the spliced out intron. To further demonstrate t
he importance of these spliceosomes to the splicing process, Brody a
nd Abelson tried to form spliceosomes with a mutant pre-mRNA that
had an A→C mutation at the branch point that rendered it unspliceabl
e. This RNA was severely impaired in its ability to form spliceosome
s. Sharp and his colleagues isolated spliceosomes from human (HeL
a) cells, also in 1985, and showed that they sedimented at 60S.
• Spliceosomes contain the pre-mRNA, of course, but they also c
ontain many RNAs and proteins. Some of these RNAs and prot
eins come in the form of small nuclear ribonucleoproteins (snR
NPs, pronounced “snurps”), which consist of small nuclear R
NAs (snRNAs) coupled to proteins. The snRNAs can be resolv
ed by gel electrophoresis into individual species designated U1
, U2, U4, U5, and U6. All fi ve of these RNAs join the spliceos
ome and play crucial roles in splicing. In principle, the consens
us sequences at the ends and branchpoint of an intron could be
recognized by either proteins or nucleic acids. We now have ex
cellent evidence that both snRNAs and protein splicing factors
are the agents that recognize these splicing signals.
• Recognition of a typical mammalian pre-mRNA intron by RNAs and
proteins. The capital letters represent bases that are well conserved, an
d the lowercase letters represent less conserved bases. Y stands for bot
h pyrimidines, R stands for both purines, and N is any base. U1 snRN
P recognizes the 5’-splice site fi rst, and then is replaced by U6 snRNP
. U2 snRNP recognizes the branchpoint, and the protein U2AF (U2-as
sociated factor) recognizes the 3’-splice site. U5 snRNP binds to the
5’- and 3’-splice sites after initial recognition by other factors.
 Capping

• Caps are made in steps: First, an RNA triphosphatase removes th

e terminal phosphate from a pre-mRNA; next, a guanylyl transfe
rase adds the capping GMP (from GTP). Next, two methyltransf
erases methylate the N7 of the capping guanosine and the 2’-O-
methyl group of the penultimate nucleotide. These events occur
early in the transcription process, before the chain length reaches
30 nt
• Cap 1 is the same as the cap shown in Figure 15.3. Cap 2 has an
other 2’-O-methylated nucleotide (two in a row). And cap 0 has
no 2’-O-methylated nucleotides. Cap 2 is found only in eukaryot
ic cells, cap 1 is found in both cellular and viral RNAs, and cap
0 is found only in certain viral RNAs. Most of the snRNAs have
another kind of cap, which contains a trimethylated guanosine.
• Reovirus cap structure (cap 1), highli
ghting thecharges. The m7G (blue gua
nine with red methyl group) contribute
s a positive charge, the triphosphate lin
kage contributes three negative charges
, the phosphodiester bond contributes o
ne negative charge, and the terminal ph
osphate contributes two negative charg
es. The net charge is therefore about -5.
The 2’-hydroxyl group on the ribose att
ached to the Y base would be methylat
ed in cap 2.
• Caps are made in steps: First, an RNA triphosphatase remo
ves the terminal phosphate from a pre-mRNA; next, a guan
ylyl transferase adds the capping GMP (from GTP). Next, t
wo methyltransferases methylate the N7 of the capping gu
anosine and the 29-O-methyl group of the penultimate nucl
eotide. These events occur early in the transcription proces
s, before the chain length reaches 30 nt.
• Sequence of events in capping. (a) RNA triph
osphatase cleaves the γ-phosphate from the
5’-end of the growing RNA. (b) Guanylyl tra
nsferase adds the GMP part of GTP (blue) to
form a triphosphate linkage, blocking the 5’-
end of the RNA. (c) A methyltransferase
• adds a methyl group (red) from AdoMet to th
e N7 of the blocking guanine. (d) Another me
thyltransferase adds a methyl group
• (red) from AdoMet to the 29-hydroxyl group
of the penultimate nucleotide. The product is
cap 1. To form a cap 2, the next nucleotide
(Y) would be methylated in a repeat of step
(d). (e) The origin of the phosphates in the tri
phosphate linkage. The α- and β- hosphates f
rom the initiating nucleotide (XTP) are highli
ghted in green, and the α-phosphate from the
capping GTP is highlighted in yellow.
 Functions of Caps

• The cap provides: (1) protection of the mRNA from degrad

ation; (2) enhancement of the mRNA’s translatability; (3) t
ransport of at least some RNAs out of the nucleus; and (4)
proper splicing of the pre-mRNA.
 Polyadenylation

• We have already seen that hnRNA is a precursor to mRNA

. One fi nding that suggested such a relationship between t
hese two types of RNA was that they shared a unique struc
ture at their 39-ends: a long chain of AMP residues called
poly(A). Neither rRNA nor tRNA has a poly(A) tail. The p
rocess of adding poly(A) to RNA is called polyadenylation
. Let us examine first the nature of poly(A) and then the po
lyadenylation process.
• James Darnell and his coworkers performed much of the early work on poly
(A) and polyadenylation. To purify HeLa cell poly(A) from the rest of the m
RNA molecule, Diana Sheiness and Darnell released it with two enzymes: R
Nase A, which cuts after the pyrimidine nucleotides C and U, and RNase T1,
which cuts after G nucleotides. In other words, they cut the RNA after every
nucleotide except the A’s, preserving only pure runs of A’s. Next, Sheiness a
nd Darnell electrophoresed the poly(A)s from nuclei and from cytoplasm to
determine their sizes. The follow figure shows the results, which demonstrat
e that both poly(A)s have major peaks that electrophoresed more slowly than
5S rRNA, at about 7S. Sheiness and Darnell estimated that this corresponde
d to about 150–200 nt. The poly(A) species observed in this experiment wer
e labeled for only 12 min, so they were newly synthesized. Little difference i
n size between these fresh nuclear and cytoplasmic poly(A)s is noticeable. H
owever, cytoplasmic poly(A) is subject to shortening. Now that poly(A)s fro
m many different organisms have been analyzed, we see an average size of f
resh poly(A) of about 250 nt.
• Size of poly(A). Sheiness and Darnell isolated radioactively labeled h
nRNA from the nuclei (blue), and mRNA from the cytoplasm (red) of
HeLa cells, then released poly(A) from these RNAs by RNase A and
RNase T1 treatment. They electrophoresed the poly(A)s, collected fra
ctions, and determined their radioactivities by scintillation counting. T
hey included 4S tRNA and 5S rRNA as size markers. Both poly(A)s el
ectrophoresed more slowly than the 5S marker, corresponding to mole
cules about 200 nt long.
• It is apparent that the poly(A) goes on the 3’-end of the m
RNA or hnRNA because it can be released very quickly wi
th an enzyme that degrades RNAs from the 39-end inward.
Furthermore, complete RNase digestion of poly(A) yielded
one molecule of adenosine and about 200 molecules of A
MP. The follow figure demonstrates that this requires poly
(A) to be at the 39-end of the molecule. This experiment al
so reinforced the conclusion that poly(A) is about 200 nt lo
ng.
• Finding poly(A) at the 39-end of hnRNA and mRNA. (a) Interior poly(A). If po
ly(A) were located in the interior of an RNA molecule, RNase A and RNase T1
digestion would yield poly(A) with a phosphate at the 39-end, then base hydrol
ysis would give only AMP. (b) Poly(A) at the 39-end of hnRNA and mRNA. Be
cause poly(A) is located at the 39-end of these RNA molecules, RNase A and T
1 digestion yields poly(A) with an unphosphorylated adenosine at the 39-end. B
ase hydrolysis gives AMP plus one molecule of adenosine. In fact, the ratio of A
MP to adenosine was 200, suggesting a poly(A) length of about 200 nt.
• We also know that poly(A) is not made by transcribing DN
A because genomes contain no runs of T’s long enough to
encode it. In particular, we fi nd no runs of T’s at the ends
of any of the thousands of eukaryotic genes that have been
sequenced. Furthermore, actinomycin D, which inhibits D
NA-directed transcription, does not inhibit polyadenylation
. Thus, poly(A) must be added posttranscriptionally. In fact
, there is an enzyme in nuclei called poly(A) polymerase (P
AP) that adds AMP residues one at a time to mRNA precur
sors.
• We know that poly(A) is added to mRNA precursors because it
is found on hnRNA. Even specific unspliced mRNA precursors
(the 15S mouse globin mRNA precursor, for example) contain
poly(A). However, as we will see later in this chapter, splicing
of some introns in a premRNA can occur before polyadenylati
on. Once an mRNA enters the cytoplasm, its poly(A) turns ove
r; in other words, it is constantly being broken down by RNase
s and rebuilt by a cytoplasmic poly(A) polymerase.
 Functions of Poly(A)

• Protection of mRNA
• Translatability of mRNA

• Poly(A) enhances both the lifetime and translatability of m

RNA. The relative importance of these two effects seems t
o vary from one system to another. At least in rabbit reticul
ocyte extracts, poly(A) seems to enhance translatability by
helping to recruit mRNA to polysomes.
 Polyadenylation Signals

• An efficient mammalian polyadenylation signal consists of

an AAUAAA motif about 20 nt upstream of a polyadenylat
ion site in a premRNA, followed 23 or 24 bp later by a G
U-rich motif, followed immediately by a U-rich motif. Ma
ny variations on this theme occur in nature, which results i
n variations in effi ciency of olyadenylation. Plant polyade
nylation signals also usually contain an AAUAAA motif, b
ut more variation is allowed in this region than in an anima
l AAUAAA. Yeast polyadenylation signals differ even mor
e, and rarely contain an AAUAAA motif.
 Poly(A) Polymerase
• Cloning and sequencing cDNAs encoding calf thymus poly(A) poly
merase reveal a mixture of 5 cDNAs derived from alternative splici
ng and alternative polyadenylation. The structures of the enzymes p
redicted from the longest of these sequences include an RNA-bindi
ng domain, a polymerase module, two nuclear localization signals,
and a serine/threonine-rich region. The latter region, but none of the
rest, is dispensable for activity in vitro.

• Poly(A) turns over in the cytoplasm. RNases tear it down, and poly
(A) polymerase builds it back up. When the poly(A) is gone, the m
RNA is slated for destruction.
 Self-study
• Other RNA Processing Events and Post-Transcriptional Co
ntrol of Gene Expression
Ribosomal RNA Processing
Transfer RNA Processing
Trans-Splicing
RNA Editing
Post-Transcriptional Control of Gene Expression:
mRNA Stability
Post-Transcriptional Control of Gene Expression:
RNA Interference
Translation Repression, mRNA Degradation, and
P-Bodies
Piwi-Interacting RNAs and Transposon Control