Chen Weiwen

Department of Biochemistry and Molecular Biology，school of basic medical sciences,Shandong University

All pictures of this PPT are from the network Chapter 14 Biosynthesis of RNA 1．About ，which of the following is TRUE?

A．use DNA as template

B．use RNA as template

C．catalyzed by DNA pol

D．use dNTPs as precursor

E．take place in cytoplasm 2. The initiation of transcription requires an RNA polymerase binding to DNA at_____

A．CAAT box

B．

C．

D．HRE

E． RNA Synthesis  Transcription: DNA –dependent RNA Synthesis

A T G C

A U G C

 RNA replication: RNA –dependent RNA Synthesis (RNA virus)

Section I Templates & Enzymes in prokaryotic transcription ① Materials involving in transcription:  Substrate: NTPs (ATP, GTP, CTP, UTP)  Template: DNA  Enzyme: DNA dependent RNA polymerase (RNA pol)  Others: protein factors, Mg2+ , Zn2+ ②RNA synthesis in the 5’→3’ direction

③NMPs are linked by 3’,5’ – phosphodiester bonds to form RNA molecule I Template for transcription in prokaryotes 1. Only one of the two DNA strands serves as a template. 2. The strand that serves as template for RNA synthesis is called the template strand. 3. The DNA strand complementary to the template strand is called the nontemplate strand/ coding strand. By convention, DNA sequences are shown as they exist in the nontemplate strand, with the 5’ terminus on the left. II RNA Is Synthesized by RNA Polymerases 1. Action mode of RNA polymerases

①Chemical mechanism of RNA synthesis ②chemical reaction formula

③Each in the newly formed RNA is selected by Watson-Crick base-pairing interactions ④Does not require a primer to initiate synthesis.

⑤RNA pol requires DNA for activity and is most active when bound to a ds DNA.

⑥In combination with a specific sequence of DNA, promoters, RNA synthesis can be initiated，but only one of the two DNA strands serves as a template. 2. Structure of the RNA polymerase (E. coli)

 a large, complex enzyme

 core enzyme: α2ββ’ω  Core enzyme has the ability to synthesize RNA on a DNA template, but it cannot recognize promoters.

 holoenzyme: α2ββ’ ωσ  σ binds transiently to the core and directs the enzyme to specific binding sites on the DNA  The holoenzyme exists in several forms, depending on the type of σ Structure of the RNA polymerase core enzyme of E. coli

 The most common subunit is σ70  the promoters of the heat shock genes are recognized and bound with by σ32  Rifampicin inhibits bacterial RNA synthesis by binding to the β subunit, preventing the promoter clearance.

Ⅲ RNA Synthesis Begins at Promoters

Asymmetric transcription

 RNA is transcribed from only one of the DNA strands that serves as the template  The template strand of different genes is not always on the same strand of DNA

 Only part of the DNA sequence is transcribed The importance of RNA polymerase orientation The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). Prokaryotic transcription units are called

 A cluster of bacterial genes can be transcribed from a single promoter.  RNA pol binds to promoters, which direct the transcription of adjacent segments of DNA (genes) How to determine the sequence of the promoter?

 RNA Polymerase Leaves Its Footprint on a Promoter  Footprinting: a technique derived from principles used in DNA sequencing, identifies the DNA sequences bound by a particular protein. RNA polymerase protection method Footprint analysis of the RNA polymerase –binding site on a DNA fragment

Footprinting results of RNA polymerase binding to the lac promoter By convention, DNA sequences are shown as they exist in the nontemplate strand, with the 5’ terminus on the left. are numbered from the transcription start site (TSS), with positive numbers to the right (in the direction of transcription) and negative numbers to the left. Prokaryotic promoters contain consensus sequences

 Analyses and comparisons of the most common class of bacterial promoters (σ70) have revealed similarities in two short sequences centered about positions -10 and -35  are important interaction sites for the σ70 subunit Consensus nucleotide sequence for the major class of E. coli promoters A sequence logo displaying the information of consensus sequence  The consensus sequence at the -10 region is (5’)TATAAT(3’).

 The consensus sequence at the -35 region is (5’)TTGACA(3’).

 A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between positions -40 and -60 in the promoters of certain highly expressed genes.

 The UP element is bound by the α subunit of RNA polymerase. Typical E. coli promoters recognized by an RNA polymerase holoenzyme containing σ70

Pribnow box  The promoter sequence determine a basal level of expression that can vary greatly from one E. coli gene to the next.  The σ subunit is a specificity factor that mediates promoter recognition and binding.  When bound to σ32, RNA polymerase is directed to a specialized set of promoters with a different consensus sequence Section II. The Process of Transcription in Prokaryotes

Initiation Elongation Termination I. Initiation

Step 1: A closed transcription complex is formed. Step 2: An open transcription complex forms in succession. Step3: the formation of the first 3’,5’- phosphodiester bond. Step 1: A closed transcription complex is formed. Step 2: an open transcription complex form in succession.

A ~17 bp region from within the -10 to +2/+3. Step3: the formation of the first 3’,5’- phosphodiester bond.

Mostly GTP/ATP Points of transcription initiation

 Holoenzyme required

 Primer not required

 First nucleotide often is GTP/ATP: 5’- pppGpN-OH3’ II. Core enzyme elongate the RNA chain independently 1. promoter clearance

① is overcome ② The σ subunit dissociates as the polymerase enters the elongation phase. ③ transcription complex move away from the promoter.

2. Transcription bubbble

 DNA duplex unwind about 17 bp distance by core enzyme movement, forming a transcription “bubble.”  the growing 3’ end of the new RNA strand base- pairs temporarily with the DNA template to form a short hybrid RNA-DNA double helix (8 bp)  G≡C > A=T > A=U  The RNA in this hybrid duplex “peels off” shortly after its formation, and the DNA duplex re-forms.  50 nt/s

Two-dimensional image of an elongating bacterial RNA polymerase, as determined by atomic force microscopy Summerize for elongation  Core enzyme required  RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’-hydroxyl end, building RNA in the 5’→3’ direction.  The template DNA strand is copied in the 3 ’→ 5’ direction (antiparallel to the new RNA strand)  Dynamic 8 bp RNA-DNA hybrid occurs in this unwound region (transcription bubble)  The DNA is unwound ahead and rewound behind as RNA is transcribed. Ⅲ In bacteria, transcription and translation are tightly coupled

1. one gene usually is transcribed by many molecules of RNA polymerase simultaneously. 2. Each mRNA is being translated by many ribosomes simultaneously. Coupling of transcription and translation in bacteria Ⅳ E. coli has at least two classes of termination signals: one class relies on a protein factor ρ and the other is ρ- independent.

1.ρ-dependent termination

① The ρ -dependent terminators usually include a CA-rich sequence called a rut (rho utilization) element.

② The ρ protein has an ATP-dependent RNA- DNA helicase activity ③ The ρ protein associates with the RNA at rut site and migrates in the 5’→3’ direction until it reaches the transcription complex that is paused at a termination site

④ There it contributes to release of the RNA transcript

⑤ ATP is hydrolyzed by ρ protein during the termination process. ρ-dependent termination

2.ρ-independent termination

① Most ρ-independent terminators have two distinguishing features.

 First: a region that produces an RNA transcript with self-complementary sequences, permitting the formation of a hairpin structure centered 15 to 20 nucleotides before the projected end of the RNA strand.

 Second: a highly conserved string of A residues in the template strand that are transcribed into U residues near the 3’ end of the hairpin. ② When a polymerase arrives at a termination site with this structure, it pauses

③ Formation of the hairpin structure in the RNA disrupts several A=U base pairs in the RNA-DNA hybrid segment and may disrupt important interactions between RNA and the RNA polymerase, facilitating dissociation of the transcript. Palindromic DNA (or RNA) sequences can form alternative structures with intrastrand base pairing. termination signal sequence contains an inverted repeat followed by a stretch of AT base pairs (top). The inverted repeat, when transcribed into RNA, can generate the secondary structure in the RNA transcript (bottom). Formation of this RNA hairpin causes RNA polymerase to pause. ρ-independent termination

The transcription cycle of bacterial RNA polymerase Section III The Biosynthesis of Eukaryote RNA I. Eukaryotic Cells At Least Have Three Kinds of Nuclear RNA Polymerases

1. Pol II transcribes most genes, including all those that encode proteins. 2. Pol II has many structural similarities to bacterial RNA pol

Prokaryote Prokaryote & Eukaryote 3. Pol II (yeast) is a huge enzyme with 12 subunits (RPB1-12)

4. The largest subunit RPB1 has an carboxyl- terminal domain (CTD) ① CTD: a long tail consisting of many repeats of a consensus heptad amino acid Tyr-Ser-Pro-Thr- Ser-Pro-Ser ② The CTD has many important roles in Pol II function through its reversible phosphorylation ③ Phosphorylation of CTD allows multi-sets of proteins to associate with it that function in transcription elongation and RNA processing. Reversible phosphorylation of CTD CTD functions like the flight deck of aircraft carrier 5. Pol Ⅱ bind to Type Ⅱ 6. There are several important differences in the way in which the bacterial and eukaryotic RNA pol function

initiation must take place on DNA that is packaged into and higher- order forms of chromatin structure , features that are absent from bacterial chromosomes.

 While bacterial RNA pol requires only a single σ factor to begin transcription, polⅡrequire many such factors, collectively called the general transcription factors. II. Cis-acting elements and transcription factors play important roles in the initiation of eukaryotic transcription Assembly of RNA Polymerase and Transcription Factors at a Promoter

DNA: promoter (cis-acting element) DNA-Pr interaction Pol II Pr General transcription factors 1. cis-acting element: the specific DNA sequences in eukaryotic genome regulating itself gene expression, including promotor, enhancer, silencer, etc.

initiator 2. Protein  Pol Ⅱ: requires an array of other proteins, called transcription factors, in order to form the active transcription complex.  : In eukaryotes, a protein that affects the regulation and transcription initiation of a gene by binding to a (cis-acting element) near or within the gene and interacting with RNA polymerase and/or other transcription factors.  general (or basal) transcription factors: required at every Pol II promoter for formation of PIC by binding to Pol II directly or indirectly  pol I – TF I; pol II- TF II ; pol III-TF III. TF IID be in common use. The General Transcription Factors Needed for Transcription Initiation by Eukaryotic RNA Polymerase II

Name Number of Function(s) subunits TBP 1 Specifically recognizes the TATA box TFIIA 2 Stabilizes binding of TFIIB and TBP to the promoter TFIIB 1 Binds to TBP; recruits Pol II–TFIIF complex; accurately positions pol II at the TSS; TFIID 13~14 Required for initiation at promoters lacking a TATA box TFIIE 2 Recruits TFIIH; regulate its ATPase and helicase activities TFIIF 2~3 Binds tightly to Pol II; binds to TFIIB and prevents binding of Pol II to nonspecific DNA sequences TFIIH 10 Unwinds DNA at TSS (helicase activity); phosphorylates CTD at Ser5 ; releases Pol II from the promoter; recruits nucleotide-excision repair proteins

TBP: TATA-binding protein TFIID: composed of TBP and 13~14 TAFs (TBP-associated factors) Three- dimensional structure of TBP (TATA-binding protein) bound to DNA The unique DNA bending caused by TBP—kinks in the double helix separated by partly unwound DNA— is thought to serve as a landmark that helps to attract the other general transcription factors BRE: A minimal pol II promoter may have a TATA box ~25 bp upstream of the Inr

3. The process of initiation

①Step 1: Formation of a closed complex （PIC）

 TBP binds to the TATA box  TBP is bound in turn by the transcription factor TFIIB, which also binds to DNA on either side of TBP.  TFIIA binds, and along with TFIIB helps to stabilize the TBP-DNA complex.  the TFIIB-TBP complex is next bound by TFIIF- Pol II complex  Finally, TFIIE and TFIIH bind to create the closed complex. Formation of preinitiation complex, PIC The eukaryotic basal transcription complex ② Step2: Creating of a open complex

TFIIH has multiple subunits and includes a DNA helicase activity that promotes the unwinding of DNA near the RNA start site (a process requiring the hydrolysis of ATP), thereby creating an open complex ③ Step3: RNA Strand Initiation and Promoter Clearance

 TFIIH phosphorylates Pol II at many places in the CTD by its kinase activity.  This causes a conformational change in the overall complex, initiating transcription.  During synthesis of the initial 60 to 70 nt, first TFIIE and then TFIIH is released, and Pol II enters the elongation phase of transcription. Transcription at RNA polymerase II promoters 4. Pol II also requires other transcription factors and protein factors.

① classes

Specific transcription factor: e.g. activator

Co-activator: e.g. mediator, TAFs Protein complex As intermediaries between the transcription activators and the Pol II complex Regulate transcription

Mediator

 consisting of 20 to 30 or more polypeptides in a protein complex

 A major eukaryotic coactivator

② Interaction: piecing theory

20000-25000 gene About 300 kinds of TF

Combinatorial control allows specific regulation of many genes using a limited repertoire of regulatory proteins. The DNA-binding sites for regulatory proteins are often inverted repeats of a short DNA sequence

homodimer homodimer heterodimer The advantages of combinatorial control The PEP carboxykinase promoter region, showing the complexity of regulatory input to this gene. III Elongation 1. Eukaryotes transcriptional elongation without the translation at same time like in prokaryotes. 2. Eukaryotic and prokaryotic transcriptional elongation follows the same basic rules

Prokaryotic transcription Pol II core enzyme complex 3. Transcription Elongation in Eukaryotes Requires Accessory Proteins

 Pol II must also contend with chromatin structure as they move along a DNA template

chaperones help by partially disassembling nucleosomes in front of a moving RNA polymerase and assembling them behind

4. RNA polymerases make about one mistake for every 104 nucleotides copied into RNA IV. The transcription of pre-mRNA is terminated and modified at 3’end at the same time. 1. Termination signal

 The pol Ⅱ ceases RNA synthesis within multiple sites located in rather long “ regions.”

 The site of cleavage/polyadenylation is flanked by two cis-acting signals: an upstream AAUAAA motif, which is usually located 11 to 30 nucleotides from the site, and a downstream U-rich or GU-rich element. Consensus nucleotide sequences that direct cleavage and polyadenylation to form the 3ʹ end of a eukaryotic mRNA 2. Process of termination Transcription at RNA polymerase II promoters Section IV The Processing and Degradation of Eukaryotic RNA

I. Processing of hn-RNA/pre-mRNA to produce mature-mRNA II. Eukaryotic rRNAs Also Undergo Processing

Ⅲ. Eukaryotic tRNAs Also Undergo Processing

Ⅳ. mRNA degradation I Processing of hn-RNA/pre-mRNA to produce mature-mRNA Key Concept

Split gene: eukaryotic structure gene is discontinuous. It is broken up into small pieces of coding sequence (expressed sequences or exons) interspersed with much longer intervening sequences or introns. Exon & Intron Exon: segments of eukaryotic gene (or of the primary transcript of that gene) that code for RNA and amino acid sequence of a polypeptide chain, and are saved as part of a functional, mature mRNA, rRNA or tRNA molecules,etc. Intron : the intervening sequences between exons of eukaryotic gene (or of the primary transcript of that gene) that are removed by splicing during RNA processing and are not included in the mature, functional mRNA, rRNA, or tRNA, etc. Overview of the processing of a eukaryotic mRNA

(hnRNA) Defining the structure of the chicken ovalbumin gene by hybridization

鸡卵清蛋白成熟mRNA与DNA杂交电镜图 NOTE!

 The number of introns equals one less than the number of exons in each gene.  Few bacterial genes contain introns.  In higher eukaryotes, the typical gene has much more intron sequence than sequences devoted to exons.  Genes for seem to have no introns.  In most cases the function of introns is not clear. Structures of 3 human genes showing the arrangement of exons and introns

Formation of the primary transcript and its processing during maturation of mRNA in a eukaryotic cell Eukaryotic RNA polymerase II as an “RNA factory.”

Tyr-Ser-Pro-Thr-Ser-Pro-Ser 1. Capping on the 5ʹ end

2. Polyadenylation of the 3ʹ end

3. Splicing: remove introns + join exons 1. Eukaryotic mRNAs Are Capped at the 5’ End

① Structure: m7Gpppm2’Npm2’Np ②Generation of the 5’ cap involves four to five separate steps. ③Occur very early in transcription, after the first 20~ 30 nt of the transcript have been added ④function

 Bind with cap-binding complex of protein，(CBC)  Prevent from degradation by the 5′ → 3′ exonucleases  Participates in exportation from the nucleus  Participates in translation initiation 2. RNA-Processing Enzymes Generate the 3ʹ End of Eukaryotic mRNAs

① Structure : 80~250 residues at 3’end

② Termination Signal：AAUAAA+CA+(GU)n

Cleavage site ③Processing CPSF：断裂和聚腺苷酸化特 CFⅠ, CFⅡ 异因子 CstF：断裂激动因子 ④ Function

 Bind with poly-A- binding proteins (PABP)  Prevent from degradation by the 3′ →5′ exonucleases  Participates in exportation from the nucleus  Participates in translation initiation Schematic illustration of an export-ready mRNA molecule and its transport through the nuclear pore.

PABPⅠ

Ⅱ 3. Pre-mRNA splicing

 DNA→hnRNA/pre-mRNA →mature-mRNA  mRNA splicing: Removal of introns and joining of exons in a primary transcript. ①Each splicing event joins two exons together while removing the intron between them as a “lariat” lariat ② The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end (splicing junction) of most introns in humans. ③ Molecular mechanism of splicing: ——two transesterifications

There are four classes of introns

 Group I introns self-splicing  Group II introns

 Spliceosomal introns: pre-mRNA, spliceosome

 tRNA introns: require ATP and an endonuclease RNA catalyze the self-splicing of some eukaryotic/prokaryotic intron RNA

1982，Thomas Cech and colleagues transcribed isolated Tetrahymena DNA (including the intron) in vitro using purified bacterial RNA pol. The resulting RNA spliced itself accurately without any protein enzymes from Tetrahymena. The discovery that could have catalytic functions was a milestone in our understanding of biological systems. Splicing mechanism of group I introns.

Coenzyme: GMP/GDP/GTP Transesterification reaction ∙∙

Splicing mechanism of group II introns.

Group Ⅰ/Ⅱ introns are self-splicing introns

 Group I introns: some nuclear, mitochondrial, and chloroplast genes that code for rRNAs, mRNAs, and tRNAs.

 Group II introns: in primary transcripts of mitochondrial or chloroplast mRNAs in fungi, algae, and plants

Self-splicing introns are ribozymes The two known classes of self-splicing intron sequences Both types of self-splicing reactions require the intron to be folded into a highly specific three dimensional structure that provides the catalytic activity for the reaction ④ Pre-mRNA Splicing Is Performed by the Spliceosome

Structure of spliceosome: snRNA U1, U2, U4, U5, U6+>100 proteins→5 types of snRNP

Mechanism Splicing mechanism in mRNA primary transcripts One of the many rearrangements that take place in the spliceosome during pre-mRNA splicing Splicing mechanism in mRNA primary transcripts The pre-mRNA splicing mechanism

Coordination of splicing and transcription brings the two splice sites together

⑤ A Gene Can Give Rise to Multiple Products by Differential RNA Processing—Alternative processing  alternative splicing: in which a particular exon may or may not be incorporated into the mature mRNA transcript.  poly(A) site choice: Complex transcripts can have more than one site for cleavage and polyadenylation  Both

The primary transcript contains molecular signals for all the alternative processing pathways. Alternative splicing

Complex poly(A) site choice transcripts

Alternative splicing + poly(A) site choice  Alternative cleavage and polyadenylation patterns The B cell receptors (BCRs) and secreted antibodies made by a B cell clone.

Regulation of the site of RNA cleavage and poly-A addition determines whether an antibody molecule is secreted or remains membrane-bound  Alternative splicing patterns

For example: Alternative splicing patterns produce, from a common primary transcript, three different forms of the myosin heavy chain at different stages of fruit fly development. Summary of splicing patterns. Alternative processing of the calcitonin gene transcript in rats

calcitonin-gene- related peptide Alternative processing of the α-tropomyosin gene from rat 4. RNA Editing Occurs at Individual Bases Can Change the Meaning of the RNA Message

 Concept: Posttranscriptional modification of an mRNA that alters the meaning of one or more codons during translation.

 Location: coding regions of primary transcripts

 Mechanism: single base alteration (mammal) by deamination reacitons.  C to U  A to I The A- to-I editing is catalyzed by ADAR enzymes.

ADAR：adenosine deaminases that act on RNA RNA腺苷脱氨酶 Mechanism of A-to-I RNA editing in mammals

双链RNA特异 性腺苷脱氨酶 A to I RNA editing occurs in GluR mRNA

 deamination of A yields I

 I preferentially pairs with C  I≈G，CAG→CGG ，Gln→ARG 2+  GluRGln: Ca pass 2+  GluRARG: Ca not pass

C-to-U editing is catalyzed by the APOBEC cytidine deaminase.

APOBEC：apoB mRNA editing catalytic peptide apoB mRNA编辑催化肽 Example：APOB gene for the apolipoprotein B component of low-density lipoprotein in vertebrates

 apoB-100 (Mr 513,000)is synthesized in the liver  apoB-48 (Mr 250,000)is synthesized in the intestine  An cytidine deaminase found only in the intestine binds to the mRNA at the 2,153 codon for Gln and converts the C to a U to create the termination codon UAA C-to-U RNA editing produces a truncated form of apolipoprotein B. II. Eukaryotic rRNAs Also Undergo Processing Electron micrograph of a thin section of a nucleolus in a human fibroblast

Transcription from tandemly arranged rRNA genes The chemical modification and nucleolytic processing of a eukaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs. Two prominent covalent modifications made to rRNA The function of snoRNAs in guiding rRNA modification. snoRNAs determine the sites of modification by base-pairing to complementary sequences on the precursor rRNA. The snoRNAs are bound to proteins to snoRNPs, which contain both the guide sequences and the enzymes that modify the rRNA. Transcription of two rRNA genes as observed under the electron microscope. Processing of pre-rRNA transcripts in vertebrates The function of the nucleolus in ribosome and other ribonucleoprotein synthesis Ⅲ. Eukaryotic tRNAs Also Undergo Processing

Processing of yeast tRNATyr in eukaryotes

and/or RNase Z cut Some modified bases of rRNAs and tRNAs, produced in posttranscriptional reactions

硫尿苷 Structure of a tRNA-splicing endonuclease docked to a precursor tRNA V. mRNA degradation 1. Deadenylation dependent mRNA decay

2.nonsense-mediated mRNA decay ——The most powerful mRNA surveillance system 3. The 5' capping process creates what type of linkage?

A. 5’-3’

B. 3’-5’

C. 5’-5’

D. 3’-3’

E. 2’-5’ 4. Splicing is the process that does which of the following?

A. Remove introns and joint exons

B. Remove exons and conserve introns

C. Remove mutated regions of primary transcript RNA

D. Add multiple adenosine bases to the end of a primary RNA transcript

E. Cleavage the 3’end 5. RNA polymerase I is the eukaryotic enzyme for

A. Trascription of rRNA

B. Trascription of tRNA

C. Trascription of mRNA

D. Trascription of snRNA

E. Trascription of snoRNA 谢 谢 观 赏