transcription in eukaryotes

1. TRANSCRIPTION
SIDRA

2. Cells contain three major types of RNA: ribosomal RNA
(rRNA), which constitutes two-thirds of the ribosomal
mass;
transfer RNA (tRNA), a set of small, compact molecules
that deliver amino acids to the ribosomes for assembly
into proteins; and
messenger RNA (mRNA), whose nucleotide sequences
direct protein synthesis. In addition, a host of other
noncoding RNA species play various roles in the
regulation of gene expression and the processing of
newly transcribed RNA molecules

3. • RNA Polymerase Resembles Other Polymerases
• The E. coli RNAP holoenzyme is an 449-kD
protein with subunit composition α2ββ’ωσ
• Once RNA synthesis has been initiated, however,
the α subunit (also called the α factor)
dissociates from the core enzyme α2ββ’ω,
• which carries out the actual polymerization
process

4. • The DNA strand that serves as a template
during transcription is known as the antisense
or noncoding strand since its sequence is
complementary to that of the RNA. The other
DNA strand, which has the same sequence as
the transcribed RNA (except for the
replacement of U with T), is known as the
sense or coding strand

5. Transcription Is Initiated at a
Promoter
• Promoters consist of 40-bp sequences that are
located on the 5’ side of the transcription start
site.
• base pair in a promoter region is assigned a
negative or positive number that indicates its
position, upstream or downstream in the
direction of RNAP travel, from the first nucleotide
that is transcribed to RNA; this start site is 1 and
there is no 0. Because RNA is synthesized in the
5¿ S 3¿ direction (see below), the promoter is
said to lie upstream of the RNA’s starting
nucleotide.

6. • Their most conserved sequence is a hexamer
the -10 position Pribnow box TATAAT
• Upstream sequences around position 35 also
have a region of sequence similarity, TTGACA.
The initiating (1) nucleotide, which is nearly
always A or G, is centered in a poorly
conserved CAT or CGT sequence

8. • rate of transcription is 20 to 50 nt/s at 37°C
(but still many times slower than the DNA
replication rate of 1000 nt/s; Section 25-2C).
The error frequency in RNA synthesis is one
wrong base incorporated for every 104
transcribed. This frequency, which is 104 to
106 times higher than that for DNA synthesis,
is tolerable because most genes are
repeatedly transcribed, because

9. • Many enzymes, particularly those involved in
basic cellular “housekeeping” functions, are
synthesized at a more or less constant rate;
they are called constitutive enzymes
• Other enzymes, termed inducible enzymes,
are synthesized at rates that vary with the
cell’s circumstances.

10. Transcription Terminates at Specific
Sites
• The transcription termination sequences of about half
of E. coli genes share two common features (Fig. 26-
10):
• 1. A series of 4 to 10 consecutive A T base pairs, with
the A’s on the template strand. The transcribed RNA is
terminated in or just past this sequence.
• 2. A G C–rich region with a palindromic sequence
that immediately precedes the series of A T’s.
• they require the action of a protein known as Rho
factor to terminate transcription

11. Eukaryotes Have Several RNA
Polymerases
• RNA polymerase I (RNAP I), which is located in
the nucleoli where ribosomes are assembled,
synthesizes the precursors of most rRNAs
• RNA polymerase II (RNAP II), which occurs in the
nucleoplasm, synthesizes the mRNA precursors.
• 3. RNA polymerase III (RNAP III), which also
occurs in the nucleoplasm, synthesizes the
precursors of 5S rRNA, the tRNAs, and a variety of
other small nuclear and cytosolic RNAs

12. • RNAP I requires a so-called core promoter
element, which spans positions -31 to +6 and
hence overlaps the transcribed region.
However, efficient transcription also requires
an upstream promoter element, which is
located between residues -187 and -107.

13. • TATA box, an AT rich sequence located 25 to
31 bp upstream from the transcription start
site. The TATA box (consensus sequence
TATAA/TAA/T resembles the -10 region of a
prokaryotic promoter (TATAAT), although it
differs in its location relative to the
transcription start site (-27 versus -10).
• For instance, many eukaryotic structural genes
have a conserved consensus sequence of
CCAAT (the CCAAT box) located between
about -70 and -90 whose alteration greatly
reduces the gene’s transcription rate.

14. • The promoters of some genes transcribed by
RNAP III are located entirely within the genes’
transcribed regions
• between nucleotides +40 and +80.

15. • Eukaryotic RNAPs, have molecular masses of
as much as 600 kD, .Each eukaryotic RNAP
contains two nonidentical “large” (>120 kD)
subunits, which are homologs of the
prokaryotic β and β’ subunits, and an array of
up to 12 different “small” (<50 kD) subunits,
two of which are homologs of the prokaryotic
α subunit and one of which is a homolog of
the ω subunit. Five of the small subunits,
including the ω homolog, are identical in all
three eukaryotic enzymes, and the α
homologs are identical in RNAPs I and III.

16. • RNAP II binds two Mg2 ions at its active site in
the vicinity of five conserved acidic residues,
which suggests that RNAPs catalyze RNA
elongation via a two-metal ion mechanism
similar to that employed by DNA polymerases
• the surface of RNAP II is almost entirely
negatively charged except for the DNA-binding
cleft and the region about the active site,
which are positively charged.

17. • RNAP II’s contain Rpb1 subunit the homolog
of the β’ subunit in prokaryotic RNAPs, has an
extraordinary C-terminal domain (CTD). In
mammals, the CTD contains 52 highly
conserved repeats with the consensus
sequence Pro-Thr-Ser-Pro-Ser-Tyr-Ser (26
repeats in yeast.
• 50 Ser residues in this hydroxylrich protein
segment are subject to reversible
phosphorylation by CTD kinases and CTD
phosphatases.

18. general transcription factors (GTFs
• Protein factors bind selectively to the
promoter regions of DNA. With class II
promoters (those transcribed by RNAP II), a
complex of at least six general transcription
factors operates as a formal equivalent of a
prokaryotic factor.

20. • TF (for transcription factor)
• preinitiation complex (PIC).

21. PIC Formation Often Begins with
TATA-Binding Protein Binding to the
TATA Box
• The first transcription factor to bind to TATA box–
containing promoters is the TATA-binding protein
(TBP), which as its name indicates, binds to the
TATA box and thereby helps identify the
transcription start site. TBP is subsequently joined
on the promoter by additional subunits to form, in
humans, the ~1122-kD, 17-subunit complex TFIID.
• The highly conserved C-terminal domain of TBP
contains two ~40% identical direct repeats of 66
residues separated by a highly basic segment

22. • The TBP, which undergoes little
conformational change on binding DNA, does
so via hydrogen bonding and van der Waals
interactions. The kinked and partially
unwound DNA is stabilized by a wedge of two
Phe side chains on each side of the saddle
structure that pry apart the two base pairs
flanking each kink from their minor groove
sides. The bent conformation of DNA creates a
stage for the assembly of other proteins to
form the PIC.

23. TFIIA, TFIIB, and TAFs Interact with
TBP and RNAP II
• The PIC requires, at a minimum, TBP, TFIIB,
TFIIE, TFIIF, and TFIIH. TFIIB consists of two
domains, an N-terminal domain (TFIIBN),
which interacts with RNAP II, and a C-terminal
domain (TFIIBC), which binds DNA and
interacts with TBP.
• Initiator element YYA+1NA/TYY

25. • The three proteins bind to the DNA just
upstream from the transcription start site,
leaving ample room for additional proteins
and RNAP II to bind. Since the
pseudosymmetric TBP has been shown to bind
to the TATA box in either orientation, it
appears that base-specific interactions
between TFIIB and the promoter function to
position TFIIB to properly orient the TBP on
the promoter.

26. • The remaining components of TFIID, which are known
as TBP-associated factors (TAFs), form a horseshoe-shaped
complex to which TFIIA and TFIIB are bound.
• In the final steps of PIC formation (Fig. 26-17), TFIIF
recruits RNAP II to the promoter in a manner
reminiscent of the way that σ factor interacts with
bacterial RNAP. In fact, the second largest of TFIIF’s
three subunits is homologous to σ 70, the predominant
bacterial σ factor, and, moreover, can specifically
interact with bacterial RNAPs (although it does not
participate in promoter recognition). Finally, TFIIE and
TFIIH join the assembly. Once this complex has been
assembled, the ATP-dependent helicase activity of
TFIIH induces the formation of the open complex so
that RNA synthesis can commence.

27. Promoters That Lack a TATA Box Also
Bind TBP
• Since the TATA-binding protein is a component of TFIID,
a general transcription factor for RNAP II. In many
cases, the presence of the Inr element is sufficient to
direct RNAP II to the correct start site. These systems
require the participation of many of the same GTFs that
initiate transcription from TATA box–containing
promoters. Surprisingly, they also require TBP. This
suggests that with TATA-less promoters, Inr recruits
TFIID such that its component TBP binds to the -30
region in a sequence-nonspecific manner.
• TBP Is a Universal Transcription Factor

28. Elongation Requires Different
Transcription Factors
• After RNAP II initiates RNA synthesis and
successfully produces a short transcript, the
transcription machinery undergoes a
transition to the elongation mode. The switch
appears to involve displacement of the finger
domain of TFIIB, which would otherwise clash
with the growing RNA chain in the active site,
as well as phosphorylation of the C-terminal
domain (CTD) of RNAP II’s Rpb1 subunit

29. • Phosphorylated RNAP II releases some of the
transcription-initiating factors and advances
beyond the promoter region. In fact, when
RNAP II moves away from (“clears”) the
promoter, it leaves behind some GTFs,
including TFIID. These proteins can reinitiate
transcription by recruiting another RNAP II to
the promoter. Consequently, the first RNAP to
transcribe a gene may act as a “pioneer”
polymerase that helps pave the way for
additional rounds of transcription

30. • During elongation, a six-protein complex
called Elongator binds to the phosphorylated
CTD of Rpb1, taking the place of the jettisoned
transcription factors. Although Elongator is
not essential for transcription by RNAP II in
vitro, its presence accelerates transcription.
Interestingly, TFIIF and TFIIH remain
associated with the polymerase during
elongation

31. Eukaryotes Lack Precise Transcription
Termination Sites.
• The sequences signaling transcriptional
termination in eukaryotes have not been
identified.
• This is largely because the termination process is
imprecise; that is, the primary transcripts of a
given structural gene have heterogeneous 3’
sequences.
• However, a precise termination site is not
required because the transcript undergoes
processing that includes endonucleolytic cleavage
at a specific site