Chapter 5 Transcription A. Transcription in prokaryotes

Chapter 5

Transcription

A. Transcription in prokaryotes

5.1 Basic principles of transcription5.1 Basic principles of transcription

5.2 Escherichia coli RNA polymerase5.2 Escherichia coli RNA polymerase

5.3 The E. coli 70 promoter5.3 The E. coli 70 promoter

5.4 transcription process. 5.4 transcription process.

An overview, the process of RNA synthesis ( initiation, elongation, termination)

Properties, subunit, subunit, ’ subunit, sigma () factor

Promoter, 70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency

Promoter binding, unwinding, RNA chain initiation, elongation, termination ( factor)

5.1: Basic principles of transcription

1.Transcription: an overview (comparison with replication)

2.The process of RNA synthesis: initiation, elongation, termination

5.1-1: Transcription: an overview

Key terms defined in this section (Gene VII)

Coding strand of DNA has the same sequence as mRNA.Downstream identifies sequences proceeding further in the direction of expression; for example, the coding region is downstream of the initiation codon.

Gene X

Primary transcriptm7Gppp

AAAAAn

+1

upstream downstream

mRNA

Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream of the coding region.

Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.

Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription

RNA Terminator is a sequence of DNA, represented at the end of the transcript, that causes RNA polymerase to terminate transcription.

RNA polymerases are enzymes that synthesize RNA using a DNA template (formally described as DNA-dependent RNA polymerases).

Primary transcript is the original unmodified RNA product corresponding to a transcription unit.

Replication: synthesis of two DNA molecules using both parental DNA strands as templates. Duplication of a DNA molecule. 1 DNA molecule 2 DNA molecules

Transcription: synthesis of one RNA molecule using one of the two DNA strands as a template.1 DNA molecule 1 RNA molecule

Replication-synthesis of the leading strand

the same direction as the replication fork moves

Replication- Synthesis of the Okazaki fragments

Opposite to the replication fork movement

Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)

Transcription

1.RNA synthesis occurs in the 5’3’ direction and its sequence corresponds to the sense strand (coding strand).

2.The template of RNA synthesis is the antisense strand (template strand).

3.Phosphodiester bonds: same as in DNA

4.Necessary components: RNA polymerase, transcription factors, rNTPs, promoter & terminator/template

DNA 双螺旋5’-CGCTATAGCGTTTGCAGGCGTTCACGGC-3’3’-GCGATATCGCAAACGTCCGCAAGTGCCG-5’

mRNA （ RNA transcript ）5’-CGCUAUAGCGUUUGCAGGCGUUCACGGC-3’

转录

DNA 非模板链（编码链） ,（ + ）正链 或正义链）

模板链 , 负链（ - ）或反义链

5.1-2: The process of RNA synthesis

1.initiation

2.elongation

3.termination

Promoter Terminator

Transcribed region Sense strand

Antisense strandDNA

RNATranscription

+1

Fig. 2. Structure of a typical transcription unit

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Before initiation: RNA pol recognizes promoter (RNA 聚合酶识

别启动子 )

1. RNA 聚合酶结合到启动子上游附近的双链 DNA 模板；

2.沿双链 DNA 滑动，找到启动子(promoter) 序列；

3.负责识别启动子的是 RNA 聚合酶的 σ亚基；

4.真核生物的转录在启动子 (promoter)序列处首先结合转录起始因子；

Initiation (template recognition) 1. Binding of an RNA polymerase to the

dsDNA

2. Slide to find the promoter

3. Unwind the DNA helix

4. Synthesis of the RNA strand at the start site (initiation site), this position called position +1

Link

Elongation

• Covalently adds ribonucleotides to the 3’-end of the growing RNA chain.

• The RNA polymerase extend the growing RNA chain in the direction of 5’ 3’

• The enzyme itself moves in 3’ to 5’ along the antisense DNA strand.

Link

Termination

• Ending of RNA synthesis: the dissociation of the RNA polymerase and RNA chain from the template DNA at the terminator site.

• Terminator: often contains self-complementary regions which can form a stem-loop or hairpin structure in the RNA products

Ter

min

ator

str

uct

ure

5.2 Escherichia coli RNA polymerase

1.E. coli RNA polymerase

2. subunit3. subunit4. ’ subunit5.sigma () factor

5.2-1 E. coli RNA polymerase

Synthesis of single-stranded RNA from DNA template.

1. Requires no primer for polymerization2. Requires DNA for activity and is most active

with a double-stranded DNA as template.3. 5’ 3’ synthesis4. Require Mg2+ for RNA synthesis activity5. lacks 3’ 5’ exonuclease activity, and the

error rate of nucleotides incorporation is 10-4 to 10-5. Is this accuracy good enough for gene expression??

6. usually are multisubunit enzyme.

RNA polymerase(NMP)n + NTP (NMP)n+1 + PPi

E. coli polymerase

1. E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA.

2. One of the largest enzyme in the cells3. Consists of at least 5 subunits in the

holoenzyme, 2 alpha (), and 1 of beta (), beta prime (’), omega () and sigma () subunits

4. Shaped as a cylindrical channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp.

5. RNA synthesis rate: 40 nt per second at 37oC

E. coli RNA polymerase

Both initiation & elongation

Initiation only

36.5 KD

36.5 KD

151 KD

155 KD

11 KD

70 KD

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E.coli RNA 聚合酶的亚基性质和功能

亚基 基因 相对分子量 亚基数 功能

rpoA 4.0104 2 core core assemble, promoter recognition

rpoB 1.51105 1 core and ’combined

together to form catalyzed center

’ rpoC 1.55105 1 core

? 11104 1 core unknown

rpoD 7.0104 1 factor different factors recognize different promoters

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可解离的 sigma 亚基赋予 RNA 聚合酶对原核启动子的特异性

核心酶Core enzyme

全酶Holoenzyme

非特异性结合启动子，并且结合紧密

特异性结合启动子，结合程度较弱

’

+

’

8-30

RNA 聚合酶起始转录

核心酶

全酶

’

启动子-35 -10

“扫描”

’

封闭复合物

rNTPs

PPi

开放复合物 ; 起始 ’

5’pppAmRNA

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RNA 聚合酶的主要功能① 识别和结合 DNA 链上的启动子；② 能沿 DNA 双链作单向运动；③ 解开 DNA 双螺旋，转录后又恢复双螺旋；④ 能同时结合单链 DNA 和转录产物 RNA ；⑤ 按 DNA 反义链为模板选择正确的底物

NTP ，以 5’ → 3’ 方向催化磷酸二酯键的形成，合成 RNA 链；

⑥ 识别转录的终止信号；⑦ 能够与转录因子相互作用，调节转录；⑧ 能在转录受到阻遏时进行自我调整，借助

辅助因子，恢复和维持 RNA 的合成。

The polymerases of bacteriophage T3 and T7 are smaller single polypeptide chains, they synthesize RNA rapidly (200 nt/sec) and recognize their own promoters which are different from E. coli promoters.

RNA polymerase differs from organism to organism

5.2-2: subunit

E. coli polymerase: subunit • Two identical subunits in the core enzyme • Encoded by the rpoA gene • Required for assembly of the core enzyme• Plays a role in promoter recognition.

Experiment: When phage T4 infects E. coli, the α subunit is modified by ADP-ribosylation of an arginine. The modification is associated with a reduced affinity for the promoters formerly recognized by the holoenzyme.

• plays a role in the interaction of RNA polymerase with some regulatory factors

大肠杆菌 RNA 聚合酶 : 亚基

1. 在核心酶中两个亚基是相同的；

2. 亚基是由 rpoA 基因编码；

3. 对于 RNA 聚合酶核心蛋白的组装是必需的；

4. 在启动子识别上可能起着重要的作用；

5.2-3&4: and ’ subunit

1. is encoded by rpoB gene, and ’ is encoded by rpoC gene .

2. Make up the catalytic center of the RNA polymerase

3. Their sequences are related to those of the largest subunits of eukaryotic RNA polymerases, suggesting that there are common features to the actions of all RNA polymerases.

4. The subunit can be crosslinked to the template DNA, the product RNA, and the substrate ribonucleotides; mutations in rpoB affect all stages of transcription. Mutations in rpoC show that ’ also is involved at all stages.

subunit may contain two domains responsible for transcription initiation and elongation

•Rifampicin ( 利福平 ) ： has been shown to bind to the β subunit, and inhibit transcription initiation by prokaryotic RNA pol. Mutation in rpoB gene can result in rifampicin resistance.

•Streptolydigins( 利迪链菌素 ) ： resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation.

’ subunit • Binds two Zn 2+ ions and may participate in

the catalytic function of the polymerase • Hyparin ( 肝素 ) ： binds to the ’ subunit and

inhibits transcription in vitro. • Hyparin competes with DNA for binding to the

polymerase.2. ’ subunit may be responsible for binding to

the template DNA .

1. β 亚基由 rpoB 基因编码； 2. RNA 聚合酶的催化中心； Rifampicin ( 利福平 ) ：与 β 亚基结合可以

抑制转录的起始， rpoB 基因突变导致对利福平的抗性；

Streptolydigins( 利迪链菌素 ) ：抗性突变也定位于 rpoB 基因，它抑制转录延伸，但不抑制起始；

3. β 亚基可能含有两个结构域负责转录的起始和延伸；

大肠杆菌 RNA 聚合酶 : 亚基

1. 亚基由 rpoC 基因编码；2. 与两个 Zn2+ 离子结合参与 RNA 聚合酶

的催化功能； • Heparin ( 肝素 ) ：在体外与 β’ 亚基结

合并抑制转录； • Heparin 与 DNA 竞争结合 RNA 聚合酶

；3. β’ 亚基可能是负责与 DNA 模板的结合；

大肠杆菌 RNA 聚合酶 : ’ 亚基

5.2-5: Sigma () factor

1. Many prokaryotes contain multiple factors to recognize different promoters. The most common factor in E. coli is 70.

2. Binding of the factor converts the core RNA pol into the holoenzyme.

3. factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter

4. factor is released from the RNA pol after initiation (RNA chain is 8-9 nt)

5. Less amount of factor is required in cells than that of the other subunits of the RNA pol.

1. 在启动子的识别上 σ 因子至关重要，对于非特异性 DNA 位点核心酶的亲和力会降低（ 104 ），而对于相应的启动子亲和力会增加；

2. 当起始后 (RNA chain is 8-9 nt) ， σ 因子会从 RNA 聚合酶中释放出来；

3. 在细胞中 σ 因子的需要量比 RNA 聚合酶其他亚基的需要量少；

大肠杆菌 RNA 聚合酶 : 亚基

5.3: The E. coli 70 promoter5.3: The E. coli 70 promoter

1. Promoter2. 70 size3. -10 sequence4. -35 sequence5. transcription

start site6. promoter

efficiency

5.3-1: Promoter5.3-1: Promoter

1. The specific short conserved DNA sequences:2. upstream from the transcribed sequence, and

thus assigned a negative number (location) 3. required for specific binding of RNA Pol.

and transcription initiation (function) 4. Were first characterized through mutations

that enhance or diminish the rate of transcription of gene

Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.

Promoter Terminator

Transcribed region Sense strand

Antisense strandDNA

RNATranscription

+1

5.3-2,3&4: 70 promoter5.3-2,3&4: 70 promoter

• Consists of a sequence of between 40 and 60 bp• -55 to +20: bound by the polymerase• -20 to +20: tightly associated with the

polymerase and protected from nuclease digestion by DNaseΙ(see the supplemental)

• Up to position –40: critical for promoter function (mutagenesis analysis)

• -10 and –35 sequence: 6 bp each, particularly important for promoter function in E. coli

---5-8 bp--- GC T A

TTGACA TATAAT-----16-18 bp-------

+1-35 sequence -10 sequence

-10 sequence (Pribonow box)1. The most conserved sequence in 70

promoters at which DNA unwinding is initiated by RNA Pol.

2. A 6 bp sequence which is centered at around the –10 position, and is found in the promoters of many different E. coli gene.

3. The consensus sequence is TATAAT. The first two bases (TA) and the final T are most highly conserved.

4. This hexamer is separated by between 5 and 8 bp from position +1, and the distance is critical.

-35 sequence: enhances recognition and interaction with the polymerase factor

• A conserved hexamer sequence around position –35

• A consensus sequence of TTGACA

• The first three positions (TTG) are the most conserved among E. coli promoters.

• Separated by 16-18 bp from the –10 box in 90% of all promoters

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核心启动子区包括

Sextama Box ;

RNA 聚合酶的识别位点 (R site)

TTGAC (Sextama Box)

-35 区 RNA 聚合酶松散结合位点

Pribnow Box ;

TATAAT (pribnow Box)

-10 区 RNA 聚合酶紧密结合位点 (B site)

Initiation site ;

+1 RNA transcriptional startpoint (I site)

A/G

-35 (R) -10 (B) +1 (I)

RNA

It can increase to recognize RNA Polyσ

It is crucial to loose DNA helix

RNA PolyRNA Poly RNA Poly

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The best interval between -10~-35 regionThe best interval between -10~-35 region

In prokaryote, the interval between -10~-35 region is about 16~19bp. Promoter activity will be decreased while the interval is <15bp or >20bp.

RNARNA 聚合酶在聚合酶在 DNADNA 链上的构型变化链上的构型变化

1. 全酶与 DNA 接触时占据长度为 75~80bp ，从 -55 ~ +20 ；

2. 进入延伸阶段， 伴随 σ因子的释放，构象发生变化，覆盖的长度为 60 bp;

3. 当新生 RNA 链聚合 15~20bp 时，构象进一步发生转变，形成延伸反应复合物，此时覆盖的长度为30~40bp 的 DNA;

• Footprinting is a technique derived from principles used in DNA sequencing. It is used to identify the specific DNA sequences that are bound by a particular protein.

RNA Polymerase Leaves Its FootPrint on a Promoter

Supplemental material

Footprinting

Footprinting

5.3-5: Transcription start site5.3-5: Transcription start site

• Is a purine in 90% of all gene

• G is more common at position +1 than A

• There are usually a C and T on either side of the start nucleotide (i.e. CGT or CAT)

The sequences of five E. coli promoters

K3-6: promoter efficiencyK3-6: promoter efficiency

There is considerable variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold .

1. The –35 sequence constitutes a recognition region which enhances recognition and interaction with the polymerase factor.

2. The -10 sequence is important for DNA unwinding.

3. The sequence around the start site influence initiation efficiency.

4. The sequence of the first 30 bases to be transcribed controls the rate at which the RNA polymerase clears the promoter, hence influences the rate of the transcription and the overall promoter strength.

Some promoter sequence are not sufficiently similar to the consensus sequence to be strongly transcribed under normal condition, thus activating factor is required for efficient initiation.

Example: Lac promoter P lac requires activating protein, cAMP receptor protein (CRP ), to bind to a site on the DNA close to the promoter sequence in order to enhance polymerase binding and transcription initiation.

Weak promoters and activating factor

5.4 Transcription process 5.4 Transcription process 1.Promoter binding2.DNA unwinding3.RNA chain initiation4.RNA chain elongation5.RNA chain termination

( factor)

1.Promoter binding

The searching process is extremely rapidlyThe searching process is extremely rapidly

Closed complex: the initial complex of the polymerase with the base-paired promoter DNA)

Closed complex: the initial complex of the polymerase with the base-paired promoter DNA)

and –10 region

Link

• The RNA polymerase core enyzme, 2’ has a general non-specific affinity for DNA, which is referred to as loose binding that is fairly stable.

• The addition of factor to the core enzyme markedly reduces the holoenzyme affinity for non-specific binding by 20 000-fold, and enhances the holoenzyme binding to correct promoter sites 100 times.

• Overall, factor binding dramatically increases the specificity of the holoenzyme for correct promoter-binding site.

The role of factor in promoter binding

2. DNA unwinding

The initial unwinding of the DNA results in formation of an open complex with the polymerase, and this process is referred to as tight binding

+1

• It is necessary to unwind the DNA so that the antisense strand to become accessible for base pairing and RNA synthesis.

• Negative supercoiling enhances the transcription of many genes, since it facilitates unwinding. Some promoters are not.

• Exceptional example: promters for the enzyme subunits of DNA gyrase are inhibited by negative supercoiling, serving as an elegant feedback loop for DNA gyrase expression.

Negative supercoiling & unwinding

3. RNA chain initiation

+1

The polymerase initially incorporates the first two nucleotides and forms a phosphodiester bond.

Starts with a GTP or ATP

Abortive initiation

• The RNA pol. goes through multiple abortive initiations before a successful initiation, which limits the overall rate of transcription

• The minimum time for promoter clearance is 1-2 seconds (a long event, the synthesis is 40 nt/ sec)

The first 9 nt are incorporated without polymerase movement along the DNA. Afterward, there is a significant probability that the chain will be aborted.

4. RNA chain elongation

• Promoter clearance: when initiation succeeds, the enzyme releases factor and forms a ternary complex of polymerase-DNA-nascent RNA, causing the polymerase to progress along the DNA to allow the re-initiation of transcription.

Transcription bubble:

1. containing ~ 17 bp of unwound DNA region and the 3’-end of the RNA that forms a hybrid helix about 12 bp.

2. moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear.

3. The E. coli polymerase moves at an average rate of ~ 40 nt per sec, depending on the local DNA sequence.

Transcription bubbleTranscription bubble

5. RNA chain termination

1.Termination occurs at terminator DNA sequences.

2.The most common stop signal is an RNA hairpin (self-complement structure)

commonly GC-rich to favor the structure stability

3. Rho-dependent and Rho-independent Termination.

TerminatorA specific DNA sequence where the transcription complex dissociate

Rho protein () independent terminator contains:

(1) self-complementary region that is G-C rich and can form a stem-loop or hairpin secondary structure. GC-rich favouring the base pairing stability and causing the polymerase to pause.

(2) a run of adenylates (As) in the template strand that are transcribed into uridylates (Us) at the end of the RNA, resulting in weak RNA-antisense DNA strand binding.

A model for -independent termination of transcription in E. coli.

The A-U base-pairing is less stable that favors the dissociation

1. Contains only the self-complementary region

2. Requires protein for termination

3. protein binds to specific sites in the single-stranded RNA

4. protein hydrolyzes ATP and moves along the nascent RNA towards the transcription complex then enables the polymerase to terminate transcription

Rho protein () dependent terminator

RNA polymerase/transcription and DNA polymerase/replication

RNA pol DNA pol

Template dsDNA is better than ssDNA

dsDNA

Require primer No Yes

Initiation promoter origin

elongation 40 nt/ sec 900 bp/sec

terminator Synthesized RNA Template DNA

In any chromosome, different genes may use different strands as template (Fig. 25-2).

B. Transcription in Eukaryotes

原核与真核基因转录的差异1 ）只有一种 RNA 聚合酶参与原核基因的转

录，真核生物有 3 种以上的 RNA 聚合酶负责不同类型的基因转录。

2 ）转录产物差别很大，以多顺反子形式存在，真核转录产物是以单顺反子形式存在成熟的 mRNA 只占初始转录产物的一小部分。

3 ）真核的转录产物需要经过剪接，加工成熟，而原核转录产物几乎不需要加工。

4 ）原核转录产物为多顺反子，大多数真核生物的 mRNA 是单顺反子；

5 ）在原核生物细胞中，转录产物可以直接作为蛋白质合成的模板，转录 mRNA

与蛋白质的翻译相互偶联。真核生物细胞的转录是在细胞核进行的成熟后通过核孔进入细胞质在此翻译出蛋白质。

5.5 The three RNA Polymerases: characterization and function

5.6 RNA Pol I genes: the ribosomal repeat

5.7 RNA Pol III genes: 5S and tRNA transcription

5.8 RNA Pol II genes: promoters and enhancers

5.9 General transcription factors and RNA Pol II initiation

5.5 The three RNA Polymerases: characterization and function

5.5 The three RNA Polymerases: characterization and function

1.Eukaryotic RNA polymerases

2.RNA polymerase subunits

3.Eukaryotic RNA polymerase activities

4.The CTD of RNA Pol II

5.5-1: Eukaryotic RNA

polymerases

5.5-1: Eukaryotic RNA

polymerases

1.The mechanism of eukaryotic transcription is similar to that in prokaryotes.

2.A lot more proteins are associated with the eukaryotic transcription machinery, which results in the much more complicated transcription.

Main Features of eukaryotic transcription

3. Three eukaryotic polymerases transcribe different sets of genes. The activities of these polymerases are distinguished by their sensitivities to the fungal toxin -amanitin ( 鹅膏菌素 , 或鹅膏蕈碱 ).

4. In addition, eukaryotic cells contain additional RNA Pols in mitochondria and chloraplasts.

Type Location Substrate -amanitin

RNA Pol I Nucleoli Most rRNAs gene Insensitive

RNA Pol II Nucleo-plasm

All protein-coding genes and some snRNA genes

Very sensitive

RNA Pol III

Nucleo-plasm

tRNAs, 5S rRNA,

U6 snRNA and other small RNAs

Moderately sensitive

Three eukaryotic polymerases

真核 RNA 聚合酶活性Eukaryotic RNA polymerase activity

1 ） Similarities to that in prokaryotic

cells

1. Don’t require a primer

2. Synthesize RNA in a 5’ to 3’ direction.

3. RNA complementary to the antisense

template strand.

2 ） Difference to bacterial polymerases

1. There are 3 RNA polymerases

2. Require more accessory factors for binding promoter DNA & initiating transcription.

3. The C-terminus of RNA Pol II largest subunit contains a stretch of heptapeptide repeats, named as carboxyl terminal domain (CTD)

• Amino acid sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Repeated 26 x (yeast) & 52x in mouse

• Involved in polymerase phosphorylation during elongation.

4. The CTD is unphosphorylated at transcription initiation, and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter (In vitro results).

5. Because it transcribes all eukaryotic protein-coding gene, RNA Pol II is the most important RNA polymerase for the study of differential gene expression. The CTD is an important target for differential activation of transcription elongation.

5.5-2: RNA polymerase subunits

5.5-2: RNA polymerase subunits

Each eukaryotic polymerase contains 12 or more subunits.– the two largest subunits are similar to each other and to the ’ and subunits of E. coli RNA Pol. – There is one other subunit in all three RNA Pol homologous to a subunit of E. coli RNA Pol.– Five additional subunits are common to all three polymerases.– Each RNA Pol contain additional four or seven specific subunit.

5.5-3: RNA polymerase activities

5.5-3: RNA polymerase activities

1.Transcription mechanism is similar to that of E. coli polymerase (How?)

2.Different from bacterial polymerasae, they require accessory factors for DNA binding.

5.5-4: The CTD of RNA pol II

5.5-4: The CTD of RNA pol II

1.The C-terminus of RNA Pol II contains a stretch of seven amino acids that is repeated 52 times in mouse enzyme and 26 times in yeast.

2.The heptapeptide sequenc is: Tyr-Ser-Pro-Thr-Ser-Pro-Ser

3. This repeated sequence is known as carboxyl terminal domain (CTD)

4. The CTD sequence may be phosphorylated at the serines and some tyrosines

5. The CTD is unphosphorylated at transcription initiation, and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter (In vitro results).

6. Because it transcribes all eukaryotic protein-coding gene, RNA Pol II is the most important RNA polymerase for the study of differential gene expression. The CTD is an important target for differential activation of transcription elongation.

5.6 RNA Pol I genes: the ribosomal repeats

5.6 RNA Pol I genes: the ribosomal repeats

1-2: Structure of the rRNA genes 1. Ribosomal RNA genes2. Role of the necleolus

3-6:RNA Pol I promoters & binding factors3. RNA Pol I promoters4. Upstream binding factor (UBF)5. Selectivity factor 16. TBP and TAFIs

7: Other rRNA genes

5.6-1&2: Structure of the rRNA genes

1. Ribosomal RNA genes

2. Role of the necleolus

5.6-1&2: Structure of the rRNA genes

1. Ribosomal RNA genes

2. Role of the necleolus

Ribosomal RNA Genes & nucleolus

1.A copy of 18S, 5.8S and 28S rRNA genes is organized as a single transcription unit in eukayotes. A 45S rRNA transcript (~13 000 nt long) is produced during transcription, which is then processed into 18S, 5.8S and 28S rRNA.

2.Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by nontranscribed spacer squences.

A single transcription unit

Tandem array

3.Continuous transcription of multiple copies of rRNA genes by RNA Pol I is essential to produce sufficient rRNAs which are packaged into ribosomes.

4.The arrays of rRNA genes (rRNA cluster) loop together to form the nucleolus and are known as nucleolar organizer regions.

5.During active rRNA synthesis, the pre-rRNA transcripts are packaged along the rRNA genes, visualizing in the electronic microscope as “Christmas tree structures”.

Christmas Tree Structures

5.6-3~6: RNA Pol I promoters & binding factors3. RNA Pol I promoters4. Upstream binding factor (UBF)5. Selectivity factor 16. TBP and TAFIs

5.6-3~6: RNA Pol I promoters & binding factors3. RNA Pol I promoters4. Upstream binding factor (UBF)5. Selectivity factor 16. TBP and TAFIs

RNA Pol I promoters1.Generally consists of a bipartite

sequence in the region preceding the start site, including core element and the upstream control elements (UCE).

2. RNA Pol I promoters in human cells are best characterized.

• Core element: -45 to +20, sufficient for transcription initiatiation.

• UCE: -180 to -107, to increase the transcription efficiency.

• Both regions are rich in G:C, with ~85% identity.

RNA Pol I promoters in human cells

Two ancillary factors (UBF & SL1) of RNA Pol I & their roles

in transcription initiation

Upstream binding factor (UBF)

• A specific DNA-binding protein that binds to UCE, as well as a different site in the upstream of the core element, causing the DNA to loop between the two sites. (two binding sites have no obvious similarity)

• UBF is essential for high level of transcription, and low level of expression occurs in its absence.

Selectivity factor 1 (SL1)1.Does not bind to promoters by

itself 2.Binds to and stabilizes the UBF-

DNA complex.3.Interacts with the free

downstream part of the core element.

4.Recruit RNA Pol I to bind and to initiate the transcription.

Subunits of SL1

SL1 consists of 4 proteins. 1.TBP (TATA-binding protein): a

factor also required for initiation by RNA Pol II and III. A critical general factor in eukaryotic transcription that ensures RNA Pol to be properly localized at the startpoint.

2.Other three subunits are referred to as TBP-associated factors (TAFIs) that are specific for RNA Pol I transcription.

The initiation complex assembles in three stages

The initiation complex proposed in your text book

UBF

UBFRNA Pol I

TBP

TAFIs

TAFIs

TA

FIs

It is not known which representation one is more accurate.

Other rRNA genes (simple)

In a simple eukaryote, Acanthamoeba( 棘阿米巴属 ), the rRNA genes have only one control element (promoter) around 12-72 bp upstream from the transcription start site.

Simple initiation:TIF (homolog of SL-1) binds to the promoter RNA Pol I bind TIF remains bound and the RNA Pol I is released for elongation.

5. 7 RNA Pol III genes: 5S and tRNA transcription

5. 7 RNA Pol III genes: 5S and tRNA transcription

1.RNA polymerase III2.tRNA genes3.5S rRNA genes4.Alternative RNA Pol III promoters5.RNA Pol III termination

5.7-1. RNA Pol III5.7-1. RNA Pol III

1. Contains at least 16 or more subunits2. Is located in nucloplasm3. Synthesizes the precursors of 5S rRNA,

the tRNAs and other small nuclear and cytosolic RNAs

Promoters for RNA polymerase III

May consist of bipartite sequences downstream of the startpoint, with boxA separated from either boxC or boxB. Or they may consist of separated sequences upstream of the startpoint (Oct, PSE, TATA).

5.7-2. tRNA genes5.7-2. tRNA genes1.The initial transcripts of tRNA

genes need to be processed to produce the mature tRNA.

2.The transcription control regions of tRNA lies after the start site within the transcribed region. The two highly conserved control sequences are called A box (5’-TGGCNNAGTGG) and B box (5’-GGTTCGANNCC).

• A box and B box also encode important sequences in the tRNA itself, the D-loop and TC-loop.

• Therefore, the highly conserved sequence in tRNAs are also highly conserved promoter DNA sequences.

3. Two complex DNA-binding factors required for tRNA transcription initiation:

• TFIIIC---binds to both the A and B boxes, an assembly factor for positioning TFIIIB.

TFIIIB: (1) binds 50 bp upstream from the A box, but has no sequence specificity and the binding position is determined by the DNA bound TFIIIC. (2) consists of three subunits, one of which is TBP, the general initiation factor; the second is called BRF (TFIIB-related factor); and the third is called B”.

TFIIIC: A and B boxes binding and a assembly factor to position TFIIIB

TFIIIB: DNA binding and RNA Pol III recruiting

5.7-3 5S rRNA genes5.7-3 5S rRNA genes

1.Tandemly arranged in a gene cluster. (In human, there is a single cluster of around 2000 genes.)

2.Transcription control regions (promoters) are organized similar to those of tRNA, except that C box is in place of B box. C box: +81-99 bp; A box: +50-65

3. Transcription factors: (1) The C box acts as the binding site for TFIIIA. (2) TFIIIA acts as an assembly factor which allows TFIIIC to interact with the 5S rRNA promoter. (3) The A box may also stabilize TFIIIC binding. (4) TFIIIC is then bound to DNA site near +1. (5) TFIIIB and TFIIIC interact to recruit RNA Pol III to initiate transcription.

TFIIIA

TFIIIC

TFIIIB

Pol III

5.7-4 Alternative RNA Pol III promoters

5.7-4 Alternative RNA Pol III promoters

Many RNA Pol III genes also rely on upstream sequences for regulation of their transcriptione.g. U6 snRNA and Epstein-Barr virus

1.Use only regulatory genes upstream from their transcription start sites.

U6 snRNA1. The coding region contains a

characteristic A box that is not required for transcription.

2. The upstream sequence contains sequences typical of RNA Pol II promoters, including a TATA box at bases –30 to –23.

3. Shares several other transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II

Suggestion: common transcription factors can regulate both RNA Pol II and Pol III genes

5.7-5 RNA Pol III termination5.7-5 RNA Pol III terminationThe RNA polymerase can terminate transcription without accessory factors. A cluster of A residue is often sufficient for termination. Xenopus borealis terminator: 5’-GCAAAAGC-3’

5.8 RNA Pol II genes: promoters and enhancers5.8 RNA Pol II genes: promoters and enhancers

1.RNA Pol II2.Cis-acting elements• Promoters• Upstream regulatory elements• Enhancers

enhancer Initiation element

Upstream element

TATA

InrmRNA

Down-stream element

5.8-1 RNA Pol II5.8-1 RNA Pol II1. located in nucleoplasm2. catalyzing the synthesis of the

mRNA precursors for all protein-coding genes.

3. RNA Pol Ⅱ-transcribed pre-mRNAs are processed through cap addition, poly(A) tail addition and splicing.

5.8-2 Promoters5.8-2 Promoters

• Most promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription. It has a 7 bp consensus sequence 5’-TATA(A/T)A(A/T)-3’.•TBP binds to TATA box that includes an additional downstream bp.

转录起始点• 转录起始位点没有广泛的序列同源性，但第一个碱

基为腺嘌呤，而两侧是嘧啶碱基。这个区域被称为起始子（ initiator ， Inr ），序列可表示为PyPyANT/APyPy 。 Inr 元件位于 -2 ～ +4 。仅由Inr 元件组成的启动子是具有可被 RNA 聚合酶 II

识别的最简单启动子形式 • 转录起始位点与 TATA box 一起组成核心启动子；• 上游调控元件具有促进转录作用

转录起始点与核心启动子 ( class II recognized by RNA polymerase II )

     Cap site ; initiation point (+1)

Capping m7GpppA/G------70 ±---AUG---(in mRNA)

Enhancer UPE core promoter Promoter (basic factor)

1-4Kb -70 -30 +1

GC island CAAC/T box TATA box Cap

起始位点区 PyPyANT/(A)PyPy

•TATA box acts in a similar way to an E. coli promoter –10 sequence to position the RNA Pol II for correct transcription initiation. The spacing but not the sequence between the TATA box and the start site is important. Transcription starts with an adenine ~50% of the time.

Some eukaryotic genes contain an initiator element instead of a TATA box. The initiator element is located around the transcription start site.

Other genes have neither a TATA box nor an initiator element, and usually are transcribed at very low rates.

5.8-3 Upstream regulatory elements

5.8-3 Upstream regulatory elements

• The basal elements (the TATA box and initiator elements) : primarily determine the location of the startpoint, and sponsor initiation only at a rather low level.

• Upstream regulatory elements (URE) such as the SP1 box and CCAAT boxes, greatly increase the frequency of initiation. URE is located within 100-200 bp from the promoter, and plays an important role in ensuring efficient transcription.

5.8-4 Enhancers5.8-4 Enhancers

Sequence elements which can activate transcription from thousands of base pairs upstream or downstream.

General characteristics of Enhancers• Exert strong activation of

transcription of a linked gene from the correct start site.

• activate transcription when placed in either orientation with respect to linked genes

• Able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site.

• Exert preferential stimulation of the closets of two tandem promoters

5.9 General transcription factors and RNA PolⅡ initiation

5.9 General transcription factors and RNA PolⅡ initiation1.RNA Pol II basal transcription

factors2.TFIID (TBP)3. TFIIA4. TFIIB and RNA Pol binding5. Factors binding after RNA Pol.6. CTD phosphorylation by TFIIH7. The initiator complex

1. TFIID: Multiprotein Complex, including TBP, other proteins are known as TAFIIs. TBP is the only protein binds to TATA box

TBP: 1. a general transcription factor bound to DNA at the TATA box.2. a general transcription required by all 3 RNA pol.

TBP

DNA

TBP: 3. Has a saddle structure with an overall dyad symmetry.

Outer surface (with ?)

Inner surface (with ?)

TBP causes DNA bending

TBP

45o Kink

2. TFIIA• binds to TFIID• stabilizes TFIID-DNA complex• contains at least 3 subunits

3. TFIIB & RNA Pol binding• binds to TFIID•Binds to RNA Pol with TFIIF

4-1 TFIIE binding•Necessary for transcription

4-2 TFIIJ, TFIIH binding•Necessary for transcription

5. phosphorylation of the polymerase CTD by TFIIHFormation of a processive RNA polymerase complex and allows the RNA Pol to leave the promoter region.

The initiator transcription complex

For TATA-box lacking RNA Pol II promoters, TBP is recruited to the initiator element 0verlapping the start site by some DNA-binding proteins, TBP then recruit the other transcription factors and polymerase similar to TATA box gene transcription.