7

RNA Synthesis and

Processing

7 Introduction

• Regulation of gene expression allows cells

to adapt to changes in their environments.
• Regulation of gene expression is
responsible for the distinct activities of the
multiple differentiated cell types that make
up complex plants and animals.

1
7 RNA Synthesis and Processing

I. Transcription in Prokaryotes
A. RNA Polymerase and Transcription
B. Repressors and Negative Control of
Transcription
C. Positive Control of Transcription
II. Eukaryotic RNA Polymerases and General
Transcription Factors
A. Prokaryotic RNA Polymerases
B. General Transcription Factors and Initiation of
Transcription by RNA Polymerase II
C. Transcription by RNA Polymerases I and III
III. Regulation of Transcription in Eukaryotes
IV. RNA Processing and Turnover

7 Transcription in Prokaryotes

• An understanding of transcription in E. coli

has provided the foundation for studies of
the far more complex mechanisms that
regulate gene expression in eukaryotic
cells.

2
7 RNA Polymerase and Transcription

• RNA polymerase is the principal enzyme

responsible for RNA synthesis.
• Like DNA polymerase, RNA polymerase is
a complex enzyme made up of multiple
polypeptide chains.

7.1 E. coli RNA polymerase

Core polymerase: 2α, β, & β’ (-σ, sigma)

7 Transcription in Prokaryotes

• A promoter is the DNA sequence to which

RNA polymerase binds to initiate
transcription of a gene.

7.2 Sequences of E. Coli promoters

initiation

3
7 Transcription in Prokaryotes

• During elongation, the polymerase remains

associated with its template while it
continues synthesis of mRNA.
 Figure 7.4
• RNA synthesis continues until the
polymerase encounters a termination
signal, at which point transcription stops.
 Figure 7.6
• After transcription stops, the RNA is
released from the polymerase, and the
enzyme dissociates from its DNA template.

7.4 Transcription by E. coli RNA polymerase

15 bases

Elongation Core polymerase

initiation

4
7.6 Transcription termination

ρ (pho) independent
ρ dependent
Stem-and-loop
≥4U

Transcription in Prokaryotes

During elongation, the polymerase remains

associated with its template while it
continues synthesis of mRNA.
Figure 7.4
RNA synthesis continues until the
polymerase encounters a termination
signal, at which point transcription stops.
Figure 7.6
After transcription stops, the RNA is
released from the polymerase, and the
enzyme dissociates from its DNA
template.

5
7 Repressors and Negative Control of
Transcription

• Transcription can be
regulated at the
stages of both
initiation and
elongation, but most
transcriptional
regulation in
bacteria operates at
the level of
initiation. 7.7 Metabolism of lactose

7 Repressors and Negative Control of

Transcription

• The model of gene regulation was

developed on the basis of experiments
involving negative control of the lac operon.
• An operon is a group of adjacent genes
transcribed as a single mRNA.
• An operator is a regulatory sequence of
DNA that controls transcription of an
operon.
• A repressor is a regulatory molecule that
blocks transcription.

6
7.9 Negative control of the lac operon
β-galactosidase (z)
Lactose permease (y)
Transacetylase (a)

o: operator

7 Repressors and Negative Control of

Transcription
• The central principle of gene regulation
exemplified by the lactose operon is that control
of transcription is mediated by the interaction of
regulatory proteins with specific DNA
sequences.
 cis-actingcontrol elements are regulatory
sequences that affect the expression of only
linked genes on the same DNA molecule.
 trans-acting factors are transcriptional
regulatory proteins that affect the expression
of genes located on other chromosomes
within the cell.

7
7 Positive Control of Transcription

• The best-studied example of positive control

in E. coli is the effect of glucose on the
expression of genes that encode enzymes
involved in the breakdown of other sugars.
• Glucose represses the lac operon even in the
presence of the normal inducer, lactose.
– Figure 7.10
– 當G存在時，即使有L，此operon還是關閉的
– 當沒有G時，還要有L，此operon才打開

7.10 Positive control of the lac operon by glucose

• Glucose represses
the lac operon
even in the
presence of the
normal inducer,
lactose.
• Low glucose
• cAMP α
• CAP: catabolite
activator protein -60

8
7 Eukaryotic RNA Polymerases and General
Transcription Factors

• All genes are transcribed by a single core

RNA polymerase in bacteria, but
eukaryotic cells contain multiple different
RNA polymerases that transcribe distinct
classes of genes.
• Transcription in eukaryotes takes place on
chromatin rather than on free DNA.
• Regulation of chromatin structure is an
important factor in the transcriptional
activity of eukaryotic genes.

7 Eukaryotic RNA Polymerases

• Eukaryotic cells
contain three
distinct nuclear
RNA
polymerases
that transcribe
different classes
of genes.

9
7 Eukaryotic RNA Polymerases

• Although all three of

the nuclear RNA
polymerases recognize
different promoters
and transcribe distinct
classes of genes, they
share several features 7.11 Structure of yeast
in common with each RNA polymerase II
other as well as with 12-17 different subunits
bacterial RNA Nine conserved subunits
polymerase.

7 General Transcription Factors and Initiation of

Transcription by RNA Polymerase II

• mRNA
• Transcription factors are specific proteins that
are required for RNA polymerase II to initiate
transcription.
• General transcription factors are involved in
transcription from all polymerase II promoters
and therefore constitute part of the basic
transcription machinery.
 Figure 7.12
 CTD: C-terminal domain

10
7.12 Formation of a polymerase II transcription initiation complex

7 General Transcription Factors and Initiation

of Transcription by RNA Polymerase II

• The TATA box is a regulatory DNA

sequence found in the promoters of many
eukaryotic genes transcribed by RNA
polymerase II.
• The TATA-binding protein, or TBP, is a
basal transcription factor that binds directly
to the TATA box.
• TBP-associated factors, or TAFs, are
polypeptides associated with TBP in the
general transcription factor, TFIID.
 TAFs + TBP = TFIID

11
7 General Transcription Factors and Initiation
of Transcription by RNA Polymerase II

• Following recruitment of RNA polymerase

II to the promoter, the binding of two
additional factors—TFIIE and
TFIIH—completes formation of the
initiation complex.

7.13 Model of the polymerase

II transcription initiation
complex

7 General Transcription Factors and Initiation

of Transcription by RNA Polymerase II

• The Mediator is a large protein complex

that stimulates basal transcription; it also
plays a key role in linking the general
transcription factors to the gene-specific
transcription factors that regulate gene
expression.
 Figure 7.14

Promoter: General transcription factors

Enhancers: Specific transcription factors

12
7.14 RNA polymerase II/Mediator complexes
CTD: polymerase II C-terminal domain
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
27 repeats in yeast & 52 in humans

7 Transcription by RNA Polymerases I and III

• RNA polymerase I is devoted solely to the

transcription of ribosomal RNA genes,
which are present in tandem repeats.

7.15 The ribosomal RNA gene

13
7 Transcription by RNA Polymerases I and III

• The promoters of ribosomal RNA genes

span about 150 base pairs just upstream
of the transcription initiation site.

-TATA box

7.16 Initiation of rDNA transcription

UBF: Upstream binding factor; SL1: selectivity factor 1

7 Transcription by RNA Polymerases I and III

• The genes for

tRNAs, 5S rRNA,
and some of the
small RNAs
involved
5S &intRNA promoter is downstream
splicing and
protein transport
are transcribed by
polymerase III.

7.17 Transcription of RNA polymerase III genes

14
7 RNA Synthesis and Processing

I. Transcription in Prokaryotes
II. Eukaryotic RNA Polymerases and General Transcription
Factors
III. Regulation of Transcription in Eukaryotes
 cis-acting regulatory sequences: promoters &
enhancers
IV. RNA Processing and Turnover
A. Processing of Ribosomal and Transfer RNAs
B. Processing of mRNA in Eukaryotes
C. Splicing Mechanisms
D. Alternative Splicing
E. RNA Editing
F. RNA Degradation

7 Regulation of Transcription in Eukaryotes

• An important difference between

transcriptional regulation in prokaryotes
and eukaryotes results from the packaging
of eukaryotic DNA into chromatin, which
limits its availability as a template for
transcription.
• Noncoding RNAs, as well as proteins,
regulate transcription in eukaryotic cells via
modifications in chromatin structure.

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7 cis-Acting Regulatory Sequences:
Promoters and Enhancers

• Certain cis-acting sequences regulate the

expression of eukaryotic genes.
• Genes transcribed by RNA polymerase II
have core promoter elements, including
the TATA box and the Inr sequence, that
serve as specific binding sites for general
transcription factors.

7.19 A eukaryotic promoter:

Thymidine kinase gene of HSV

7 cis-Acting Regulatory Sequences:

Promoters and Enhancers
7.20 The SV40 enhancer

• Enhancers are transcriptional regulatory

sequences that can be located at a site
distant from the promoter. (Figure 7.20)
• The activity of enhancers depends on
neither their distance nor their orientation
with respect to the transcription initiation
site. (Figure 7.21)

16
7 7.21 Action of enhancers

7 7.21 Action of enhancers (Part 2)

17
7 cis-Acting Regulatory Sequences:
Promoters and Enhancers
• DNA looping allows a transcription factor
bound to a distant enhancer to interact
with proteins associated with the RNA
polymerase/Mediator complex at the
promoter.

7.22 DNA looping

7 RNA Processing and Turnover

• Transcription is usually only the beginning

of the series of events required to produce
a functional RNA.
• Primary transcripts of eukaryotic mRNAs
undergo extensive modifications before
they are transported from the nucleus to
the cytoplasm to serve as templates for
protein synthesis.

18
7 Processing of Ribosomal and Transfer
RNAs

• pre-rRNA is the primary transcript (45S)

that is cleaved to form individual ribosomal
RNAs—the 28S, 18S, and 5.8S rRNAs of
higher eukaryotic cells.
Then addition of methylation groups

7.42 Processing of ribosomal RNAs

7 Processing of Ribosomal and Transfer

RNAs
• pre-tRNA is the primary transcript that is
cleaved to form transfer RNAs. (Figure 7.43)
• RNase P is an enzyme capable of catalyzing
pre-tRNA cleavage.
• A ribozyme is an enzyme in which RNA rather
than protein is responsible for catalytic activity.

20 tRNAs

19
7.43 Processing of transfer RNAs (Part 1)

7.43 Processing of transfer RNAs (Part 2)

20
7 Processing of mRNA in Eukaryotes
• Pre-mRNA is the primary transcript that is
processed to form messenger RNA in eukaryotic
cells. (Figure 7.44)
A 7-methylguanosine cap is what is added
during the modification of the 5′ end of a
transcript.
A poly-A tail is a tract of about 200 adenine
nucleotides added to the 3′ ends of eukaryotic
mRNAs.
 Polyadenylation is the process of adding a poly-
A tail to a pre-mRNA.
 Figure 7.45

7.44 Processing of eukaryotic messenger RNAs

Special: 異於正常之磷酸雙酯鍵，
不易被分解

21
7.45 Formation of the 3’ ends of eukaryotic mRNAs

Function of 7-methylguanosine cap & poly-A tail:

保護及穩定

7 Splicing Mechanisms

• Pre-mRNA splicing proceeds in two steps.

5’ splice site

7.47 Splicing of pre-mRNA Lariat-like intron

22
7 Splicing Mechanisms

• Spliceosomes are large complexes

composed of proteins and RNAs.
• Small nuclear RNAs, or snRNAs, are
nuclear RNAs that range in size from 50 to
200 bases.
• Small nuclear ribonucleoprotein particles,
or snRNPs, are complexes of snRNAs with
proteins that play central roles in the
splicing process.

7 Splicing Mechanisms

• The first step in spliceosome assembly is

the binding of U1 snRNP to the 5′ splice of
pre-mRNA.

23
7.48 Assembly of the spliceosome

7 Splicing Mechanisms

• Self-splicing is the ability of some RNAs to

catalyze the removal of their own introns.
• Self-splicing RNAs are divided into two
classes on the basis of their reaction
mechanisms.

24
7.50 Self-splicing introns

Group I intron Group II intron

7 Splicing Mechanisms

• The SR splicing factors bind to specific

sequences within exons and act to recruit
U1 snRNPs to the 5′ splice site.

7.51 Role of splicing factors in spliceosome assembly

25
7 Alternative Splicing

• Alternative splicing is the generation of

different mRNAs by varying the pattern of
pre-mRNA splicing.
• In the sex determination of Drosophila,
alternative splicing of the same pre-mRNA
determines whether a fly is male or female.

7.52 Alternative splicing in Drosophila sex determination

26
7 RNA Editing

• RNA editing is an RNA processing event

that alters the protein-coding sequences of
some mRNAs.

4536 aa 2152 aa

7.54 Editing of apolipoprotein B mRNA

7 期中考

• 期中考 (9th wk)：

 11/11 (三) 10:20-11:30
 Chap 1~7

 70 英文選擇
 1.5 points each

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