Section III: Expression of the Genome

Section III: Expression of the Genome
Mechanisms of transcription RNA splicing Translation The genetic code

Mechanisms of Transcription
chapter12:mechanisms of transcription 2017/4/15 Molecular Biology Course Mechanisms of Transcription RNA polymerase and transcription cycle The transcription cycle in bacteria Transcription in eukaryotes

The Central Dogma DNA RNA PROTEIN Transcription Translation
replication

Transcription is very similar to DNA replication but there are some important differences:
RNA is made of ribonucleotides RNA polymerase catalyzes the reaction The synthesized RNA does not remain base-paired to the template DNA strand Less accurate (error rate: 10-4)

Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. (Replication?)

Transcription of DNA into RNA
Transcription bubble Transcription of DNA into RNA

Topic 1: RNA Polymerase and The Transcription Cycle
Mechanisms of Transcription Topic 1: RNA Polymerase and The Transcription Cycle

RNA polymerases come in different forms, but share many features
RNA polymerase and the transcription cycle RNA polymerases come in different forms, but share many features RNA polymerases performs essentially the same reaction in all cells Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III

RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genes RNA Pol I transcribe the large ribosomal RNA precursor gene RNA Pol II transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes

The subunits of RNA polymerases

The bacterial RNA polymerase
The core enzyme alone synthesizes RNA b’ a b a w

The same color indicate the homologous of the two enzymes a w
prokaryotic b’ RNAP Comparison a b The same color indicate the homologous of the two enzymes a w eukaryotic RPB2 RPB3 RPB1 RPB11 RPB6

“Crab claw” shape of RNAP (The shape of DNA pol is__)
Active center cleft

There are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzyme’s active center cleft

Transcription by RNA polymerase proceeds in a series of steps
RNA polymerase and the transcription cycle Transcription by RNA polymerase proceeds in a series of steps Initiation Elongation Termination

Initiation Promoter: the DNA sequence that initially binds the RNA polymerase The structure of promoter-polymerase complex undergoes structural changes to proceed transcription DNA at the transcription site unwinds and a “bubble” forms Direction of RNA synthesis occurs in a 5’-3’ direction (3’-end growing)

initiation Binding (closed complex) Promoter “melting” (open complex) Initial transcription

Elongation Once the RNA polymerase has synthesized a short stretch of RNA (~ 10 nt), transcription shifts into the elongation phase. This transition requires further conformational change in polymerase that leads it to grip the template more firmly. Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain

Termination After the polymerase transcribes the length of the gene (or genes), it will stop and release the RNA transcript. In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.

Elongation and termination

Transcription initiation involves 3 defined steps
RNA polymerase and the transcription cycle Transcription initiation involves 3 defined steps Forming closed complex Forming open complex Promoter escape

Closed complex The initial binding of polymerase to a promoter
DNA remains double stranded The enzyme is bound to one face of the helix

Open complex the DNA strand separate over a distance of ~14 bp (-11 to +3 ) around the start site (+1 site) Replication bubble forms

Stable ternary complex
RNA polymerase and the transcription cycle Stable ternary complex The enzyme escapes from the promoter The transition to the elongation phase Stable ternary complex =DNA +RNA + enzyme

Topic 2 The transcription cycle in bacteria
Mechanisms of Transcription Topic 2 The transcription cycle in bacteria

The transcription cycle in bacteria
Bacterial promoters vary in strength and sequences, but have certain defining features

Holoenzyme= factor + core enzyme
In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity? ,

Promoters recognized by E. coli s factor
The predominant s factor in E. coli is s70. Promoter recognized by s70 contains two conserved sequences (-35 and –10 regions/elements) separated by a non-specific stretch of nt. Position +1 is the transcription start site.

bacterial promoter The distance is conserved s70 promoters contain recognizable –35 and –10 regions, but the sequences are not identical. Comparison of many different promoters derives the consensus sequences reflecting preferred –10 and –35 regions

Consensus sequence of the -35 and -10 region

Promoters with sequences closer to the consensus are generally “stronger” than those match less well. (What does “stronger” mean?) The strength of the promoter describes how many transcripts it initiates in a given time.

bacterial promoter Confers additional specificity UP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase

bacterial promoter Another class of s70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region

The s factor mediates binding of polymerase to the promoter
The transcription cycle in bacteria The s factor mediates binding of polymerase to the promoter The s70 factor comprises four regions called s region 1 to s region 4.

regions of s Region 4 recognizes -35 element Region 2 recognizes -10 element Region 3 recognizes the extended –10 element

Binding of –35 Two helices within region 4 form a common DNA-binding motif, called a helix-turn-helix motif One helix inserts into the DNA major groove interacting with the bases at the –35 region. The other helix lies across the top of the groove, contacting the DNA backbone Helix-turn-helix DNA-binding motif

Interaction with –10 is more elaborate (精细) and less understood
The -10 region is within DNA melting region The a helix recognizing –10 can interacts with bases on the non-template strand to stabilize the melted DNA.

UP-element is recognized by a carboxyl terminal domain of the a-subunit (aCTD), but not by s factor
s and a subunits recruit RNA pol core enzyme to the promoter

This transition is called Isomerization (异构化)
The transcription cycle in bacteria Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA This transition is called Isomerization (异构化)

For s70 –containing RNA polymerase, isomerization is a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form. (No extra energy requirement)

Change of the promoter DNA
the opening of the DNA double helix, called “melting”, at positions -11 and +3.

The striking structural change in the polymerase
1. the b and b’ pincers down tightly on the downstream DNA 2. A major shift occurs in the N-terminal region of s (region 1.1) shifts. In the closed complex, s region 1.1 is in the active center; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft

channels into and out of the open complex
NTP uptake channel is in the back DNA entering channel channels into and out of the open complex

Transcription is initiated by RNA polymerase without the need for a primer Initiation requires: The initiating NTP (usually an A) is placed in the active site The initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme

RNA polymerase synthesizes several short RNAs before entering the elongation phase Abortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nt.

Structural barrier for the abortive initiation
The 3.2 region of s factor lies in the middle of the RNA exit channel in the open complex. Ejection of this region from the channel (1) is necessary for further RNA elongation, (2) takes the enzyme several attempts

channels into and out of the open complex
NTP uptake channel is in the back DNA entering channel channels into and out of the open complex

The elongating polymerase is a processive machine that synthesizes and proofreads RNA

Synthesizing by RNA polymerase
DNA enters the polymerase between the pincers Strand separation in the catalytic cleft NTP addition RNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time) DNA strand annealing in behind

Proofreading by RNA polymerase
Pyrohosphorolytic （焦磷酸键解）editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi. Hydrolytic （水解）editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. This is stimulated by Gre factor, a elongation stimulation factor.

Transcription is terminated by signals within the RNA sequence
The transcription cycle in bacteria Transcription is terminated by signals within the RNA sequence Terminators: the sequences that trigger the elongation polymerase to dissociate from the DNA Rho-dependent (requires Rho protein) Rho-independent, also called intrinsic (内在) terminator

Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs.

transcription termination
Weakest base pairing: A:U make the dissociation easier

Rho (r) -dependent terminators
Have less well-characterized RNA elements, and requires Rho protein for termination Rho is a ring-shaped single-stranded RNA binding protein, like SSB Rho binding can wrest (夺取) the RNA from the polymerase-template complex using the energy from ATP hydrolysis Rho binds to rut (r utilization) RNA sites Rho does not bind the translating RNA

the r transcription terminator
RNA tread trough the “ring” Hexamer, Open ring

Topic 3: transcription in eukaryotes
Mechanisms of Transcription Topic 3: transcription in eukaryotes

Comparison of eukaryotic and prokaryotic RNA polymerases
Eukaryotes: Three polymerase transcribes different class of genes: Pol I-large rRNA genes; Pol II-mRNA genes; Pol III- tRNA, 5S rRNA and small nuclear RNA genes (U6) Prokaryotes: one polymerase transcribes all genes

Comparison of eukaryotic and prokaryotic promoter recognition
Eukaryotes: general transcription factors (GTFs). TFI factors for RNAP I, TFII factors for RNAP II and TFIII factors for RNAP III Prokaryotes: s factors

In addition to the RNAP and GTFs, in vivo transcription also requires
Mediator complex DNA-binding regulatory proteins chromatin-modifying enzymes Why??

The transcription in eukaryotes
RNA polymerase II core promoters are made up of combinations of 4 different sequence elements Eukaryotic core promoter (~40 nt): the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro

Pol II core promoter TFIIB recognition element (BRE)
The TATA element/box Initiator (Inr) The downstream promoter element (DPE)

Regulatory sequences The sequence elements other than the core promoter that are required to regulate the transcription efficiency Those increasing transcription: Promoter proximal elements Upstream activator sequences (UASs) Enhancers Those repressing elements: silencers, boundary elements, insulators (绝缘体)

RNA Pol II forms a pre-initiation complex with GTFs at the promoter
The transcription in eukaryotes RNA Pol II forms a pre-initiation complex with GTFs at the promoter The involved GTFIIs (general transcription factor for Pol II) TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors) TFIIA, B, F, E, H

TBP in TFIID binds to the TATA box
TFIIA and TFIIB are recruited with TFIIB binding to the BRE RNA Pol II-TFIIF complex is then recruited TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex Promoter melting using energy from ATP hydrolysis by TFIIH ) Promoter escapes after the phosphorylation of the CTD tail

Promoter escape Stimulated by phosphorylation of the CTD (C-terminal domain) tail of the RNAP II CTD contains the heptapeptide repeat Tyr-Ser-Pro-Thr-Ser-Pro-Ser Phosphorylation of the CTD “tail” is conducted by a number of specific kinases including a subunit of TFIIH

TBP binds to and distorts DNA using a b sheet inserted into the minor groove Unusual (P367 for the detailed mechanism) The need for that protein to distort the local DNA structure

A:T base pairs (TATA box) are favored because they are more readily distorted to allow initial opening of the minor groove

The other GTFs also have specific roles in initiation
The transcription in eukaryotes The other GTFs also have specific roles in initiation ~ 10 TAFs: (1) two of them bind DNA elements at the promoter (Inr and DPE); (2) several are histone-like TAFs and might bind to DNA similar to that histone does; (3) one regulates the binding of TBP to DNA

TFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3)bridging TBP and the polymerase, (4) the N-terminal inserting in the RNA exit channel resembles the s3.2 . TFIIB-TBP-promoter complex

TFIIF: (1) a two subunit factor, (2) binding of Pol II-TFIIF stabilizes the DNA-TBP-TFIIB complex, which is required for the followed factor binding TFIIE: recruits and regulates TFIIH TFIIH: (1) controls the ATP-dependent transition of the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two functions as ATPase and one is protein kinase. (3) important for promoter melting and escape. (4) ATPase functions in nucleotide mismatch repair, called transcription-coupled repair.

in vivo, transcription initiation requires additional proteins
The transcription in eukaryotes in vivo, transcription initiation requires additional proteins The mediator complex Transcriptional regulatory proteins Nucleosome-modifying enzymes To counter the real situation that the DNA template in vivo is packed into nucleosome and chromatin

assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators

Mediator consists of many subunits, some conserved from yeast to human
The transcription in eukaryotes Mediator consists of many subunits, some conserved from yeast to human More than 20 subunits 7 subunits show significant sequence homology between yeast and human Only subunit Srb4 is essential for transcription of essentially all Pol II genes in vivo Organized in modules (模块)

comparison of the yeast and human mediators

Pol II + mediator + some of GTFs core polymerase + s factor
Eukaryotic RNA Pol II holoenzyme is a putative preformed complex: Pol II + mediator + some of GTFs Prokaryotic RNA Polymerase holoenzyme: core polymerase + s factor

A new set of factors stimulate Pol II elongation and RNA proofreading
The transcription in eukaryotes A new set of factors stimulate Pol II elongation and RNA proofreading

Transition from the initiation to elongation involves the Pol II enzyme shedding (摆脱) most of its initiation factors (GTF and mediators) and recruiting other factors: (1) Elongation factors: factors that stimulate elongation, such as TFIIS and hSPT5. (2) RNA processing (RNA 加工) factors Recruited to the C-terminal tail of the CTD of RNAP II to phosphorylate the tail for elongation stimulation, proofreading, and RNA processing like splicing and polyadenylation.

RNA processing enzymes are recruited by the tail of polymerase

Some elongation factors
P-TEFb: phosphorylates CTD Activates hSPT5 Activates TAT-SF1 TFIIS: Stimulates the overall rate of elongation by resolving the polymerase pausing Proofreading

Elongation polymerase is associated with a new set of protein factors required for various types of RNA processing RNA processing: Capping of the 5’ end of the RNA Splicing of the introns (most complicated) Poly adenylation (多聚腺苷化) of the 3’ end

Elongation, termination of transcription, and RNA processing are interconnected/ coupled (偶联的) to ensure the coordination (协同性) of these events Evidence: this is an overlap in proteins involving in those events The elongation factor hSPT5 also recruits and stimulates the 5’ capping enzyme The elongation factor TAT-SF1 recruits components for splicing

Function of poly(A) tail
Increased mRNA stability Increased translational efficiency Splicing of last intron AAAAAA

Function of 5´cap Protection from degradation
Increased translational efficiency Transport to cytoplasm Splicing of first intron

RNA processing 1 5’ end capping
The “cap”: a methylated guanine joined to the RNA transcript by a 5’-5’ linkage The linkage contains 3 phosphates 3 sequential enzymatic reactions Occurs early

Splicing: joining the protein coding sequences
Dephosphorylation of Ser5 within the CTD tail leads to dissociation of capping machinery Further phosphorylation of Ser2 recruits the splicing machinery

3’ end polyadenylation Linked with the termination of transcription
The CTD tail is involved in recruiting the polyadenylation enzymes The transcribed poly-A signal triggers the reactions Cleavage of the message Addition of poly-A Termination of transcription

polyadenylation and termination
1. CPSF (cleavage and polyadenylation specificity factor) & CstF (cleavage stimulation factor) bind to the poly-A signal, leading to the RNA cleavage 2. Poly-A polymerase (PAP) adds ~ 200 As at the 3’ end of the RNA, using ATP as a substrate polyadenylation and termination

What terminates transcription by polymerase?

Models to explain the link between polyadenylation and termination (see the animation on your CD)
Model 1: The transfer of the 3’ processing enzyme to RNAP II induces conformational change—RNAP II processivity reduces—spontaneous termination Model 2: absence of a 5’cap on the second RNA molecule—recognized by the RNAP II as improper—terminate transcription

RNA Pol I & III recognize distinct promoters , using distinct sets of transcription factors, but still require TBP The transcription in eukaryotes Pol I: transcribes rRNA precursor encoding gene (multi-copy gene) Pol III: transcribes tRNA genes, snRNA genes and 5S rRNA genes

Pol I promoter recognition
Upstream control element UBF binds to the upstream of UCE, bring SL1 to the downstream part of UCE. SL1 in turn recruits RNAP I to the core promoter for transcription Pol I promoter region

Pol III promoter recognition 1. Different forms, 2
Pol III promoter recognition 1. Different forms, 2. locates downstream of the transcription site TFIIIC binds to the promoter, recruiting TFIIIB, which in turn recruits RNAP III Pol III core promoter

Key points of the chapter
RNA polymerases (RNAP, 真核和原核的异同) and transcription cycle (Initiation is more complicate, details in bacteria) Transcription cycle in bacteria: (1) promoters (elements), s factor (4 domains), aCTD, abortive initiation (why?) (2) Structures accounting for formation of the closed complex, transitions to open complex and then stable ternary complex. (3) Elongation and editing by polymerase (10-4) (4) Termination: Rho-independent and Rho-dependent mechanism

Transcription cycle in eukaryotes:
Promoters (elements), general transcription factors (GTF), RNAP II transcription ---the roles of GTFs and the CTD tail of RNAP II in promoter recognition, formation of the pre-initiation complex, promoter melting, promoter escape ---in vivo requires mediator complex, nucleosome modifying enzymes and transcription regulatory proteins. ---elongation and proofreading involve a new set of GTFs (What) ---coupled with RNA processing (How)

RNAP I and III transcription
---GTFs and promoter recognition, formation of the initiation complex

Molecular Biology Course
RNA Splicing

Primary transcript

Most of the eukaryotic genes are mosaic (嵌合体), consisting of intervening sequences separating the coding sequence Exons (外显子): the coding sequences Introns (内含子) : the intervening sequences RNA splicing: the process by which introns are removed from the pre-mRNA. Alternative splicing (可变剪接): some pre-mRNAs can be spliced in more than one way , generating alternative mRNAs. 60% of the human genes are spliced in this manner.

Topic 1 : THE CHEMISTRY OF RNA SPLICING

Sequences within the RNA Determine Where Splicing Occurs
The chemistry of RNA splicing The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.

The consensus sequences for human

5’splice site (5’剪接位点): the exon-intron boundary at the 5’ end of the intron
Branch point site (分枝位点): an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

The intron is removed in a Form Called a Lariat (套马索) as the Flanking Exons are joined
The chemistry of RNA splicing Two successive transesterification: Step 1: The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5’ splice site. As a result, the 5’ exon is released and the 5’-end of the intron forms a three-way junction structure.

Three-way junction

The structure of three-way function This figure has an error

Step 2: The OH of the 5’ exon attacks the phosphoryl group at the 3’ splice site. As a consequence, the 5’ and 3’ exons are joined and the intron is liberated in the shape of a lariat.

Exons from different RNA molecules can be fused by Trans-splicing
The chemistry of RNA splicing Trans-splicing: the process in which two exons carried on different RNA molecules can be spliced together.

Trans-splicing Not a lariat

THE SPLICESOME MACHINERY
RNA Splicing Topic 2 THE SPLICESOME MACHINERY

RNA splicing is carried out by a large complex called spliceosome
The spliceosome machinery RNA splicing is carried out by a large complex called spliceosome The above described splicing of introns from pre-mRNA are mediated by the spliceosome. The spliceosome comprises about 150 proteins and 5 snRNAs. Many functions of the spliceosome are carried out by its RNA components.

The five RNAs (U1, U2, U4, U5, and U6, 100-300 nt) are called small nuclear RNAs (snRNAs).
The complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNP, pronounces “snurps”). The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reaction

Three roles of snRNPs in splicing
1. Recognizing the 5’ splice site and the branch site. 2. Bringing those sites together. 3. Catalyzing (or helping to catalyze) the RNA cleavage. RNA-RNA, RNA-protein and protein-protein interactions are all important during splicing.

RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA

Topic 3 SPLICING PATHWAYS
RNA Splicing Topic 3 SPLICING PATHWAYS

Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway (Fig. 13-8)
Splicing pathways Assembly step 1 1. U1 recognize 5’ splice site. 2. One subunit of U2AF binds to Py tract and the other to the 3’ splice site. The former subunits interacts with BBP and helps it bind to the branch point. 3. Early (E) complex is formed

Assembly step 2 1. U2 binds to the branch site, and then A complex is formed. 2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded(Figure 13-6). This A residue is available to react with the 5’ splice site.

E complex A complex

Assembly step 3 1. U4, U5 and U6 form the tri-snRNP Particle. 2. With the entry of the tri-snRNP, the A complex is converted into the B complex.

A complex B complex

Assembly step 4 U1 leaves the complex, and U6 replaces it at the 5’ splice site. U4 is released from the complex, allowing U6 to interact with U2 (Figure 13-6c).This arrangement called the C complex.

B complex C complex in which the catalysis has not occurred yet

Catalysis Step 1: Formation of the C complex produces the active site, with U2 and U6 RNAs being brought together Formation of the active site juxtaposes (并置) the 5’ splice site of the pre-mRNA and the branch site, allowing the branched A residue to attack the 5’ splice site to accomplish the first transesterfication (转酯) reaction.

Catalysis Step 2: U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3’-OH of the 5’ exon attacks the 3’ splice site. Final Step: Release of the mRNA product and the snRNPs

C complex 1st reaction 2nd reaction

splicesome-mediated splicing reactions
E complex A complex B complex C complex （没有该complex的图）

How does spliceosome find the splice sites reliably
Splicing pathways Two kinds of splice-site recognition errors Splice sites can be skipped. “Pseudo” splice sites could be mistakenly recognized, particularly the 3’ splice site.

Reasons for the recognition errors
(1) The average exon is 150 nt, and the average intron is about 3,000 nt long (some introns are near 800,000 nt) It is quite challenging for the spliceosome to identify the exons within a vast ocean of the intronic sequences.

(2) The splice site consensus sequence are rather loose
(2) The splice site consensus sequence are rather loose. For example, only AGG tri-nucleotides is required for the 3’ splice site, and this consensus sequence occurs every 64 nt theoretically.

Two ways to enhance the accuracy of the splice-site selection
1. Because the C-terminal tail of the RNA polymerase II carries various splicing proteins, co-transcriptional loading of these proteins to the newly synthesized RNA ensures all the splice sites emerging from RNAP II are readily recognized, thus preventing exon skipping.

2. There is a mechanism to ensure that the splice sites close to exons are recognized preferentially. SR proteins bind to the ESEs (exonic splicing enhancers) present in the exons and promote the use of the nearby splice sites by recruiting the splicing machinery to those sites

SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites.

SR proteins are essential for splicing
Ensure the accuracy and efficacy of constitutive splicing Regulate alternative splicing There are many varieties of SR proteins. Some are expressed preferentially in certain cell types and control splicing in cell-type specific patterns

Topic 4 ALTERNATIVE SPLICING
RNA Splicing Topic 4 ALTERNATIVE SPLICING

Single genes can produce multiple products by alternative splicing
Many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products.

Drosophila DSCAM gene can be spliced in 38,000 alternative ways

There are five different ways to alternatively splice a pre-mRNA

Alternative splicing can be either constitutive or regulated
Constitutive alternative splicing: more than one product is always made from a pre-mRNA Regulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types

An example of constitutive alternative splicing : Splicing of the SV40 T antigen RNA

Alternative splicing is regulated by activators and repressors
The regulating sequences : exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS). The former enhance and the latter repress splicing. Proteins that regulate splicing bind to these specific sites for their action Alternative splicing

SR proteins binding to enhancers act as activators.
(1) One domain is the RNA-recognition motif (RRM) (2) The other domain is RS domain rich in arginine and serine. This domain mediates interactions between the SR proteins and proteins within the splicing machinery.

hnRNPs binds RNA and act as repressors
Most silencers are recognized by hnRNP ( heterogeneous nuclear ribonucleoprotein) family. These proteins bind RNA, but lack the RS domains. Therefore, (1) They cannot recruit the splicing machinery. (2) they block the use of the specific splice sites that they bind.

Regulated alternative splicing

An example of repressors: inhibition of splicing by hnRNPI
Coats the RNA and makes the exons invisible to the splicing machinery Binds at each end of the exon and conceals (隐藏) it

The outcome of alternative splicing:
1. Producing multiple protein products, called isoforms. 2. Switching on and off the expression of a given gene. In this case, one functional protein is produced by a splicing pattern, and the non-functional proteins are resulted from other splicing patterns.

A small group of intron are spliced by minor spliceosome
Alternative splicing This spliceosome works on a minority of exons, and those have distinct splice-site sequence. The chemical pathway is the same as the major spliceosome.

U11 and U12 are in places of U1 and U2, respectively
The AT-AC spliceosome

Topic x : Self-splicing introns
RNA Splicing Topic x : Self-splicing introns 自剪接内含子

Self-splicing introns reveal that RNA can catalyze RNA splicing
Self-splicing introns: the intron itself folds into a specific conformation within the precursor RNA and catalyzes the chemistry of its own release and the exon ligation Splicing pathways

Adams et al., Nature 2004, Crystal structure of a self-splicing group I intron with both exons

Practical definition for self-splicing introns: the introns that can remove themselves from pre-RNAs in the test tube in the absence of any proteins or other RNAs. There are two classes of self-splicing introns, group I and group II self-splicing introns.

Three class of RNA Splicing
Abundance Mechanism Catalytic Machinery Nuclear pre-mRNA Very common; used for most eukaryotic genes Two transesterification reactions; branch site A Major spliceosome Group II introns Rare; some eu-Karyotic genes from organelles and prokaryotes Same as pre-mRNA RNA enzyme encoded by intron (ribozyme) Group I introns Rare; nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genes Two transesterific-ation reactions; exogenous G Same as group II introns

The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.

Group I introns release a linear intron rather than a lariat
Instead of using a branch point A, group I introns use a free G to attack the 5’ splice site. This G is attached to the 5’ end of the intron.The 3’-OH group of the 5’ exon attacks the 5’ splice site. The two-step transesterification reactions are the same as that of splicing of the group II intron and pre-mRNA introns. Splicing pathways

G instead of A a Lariat intron a linear intron

Group I introns Smaller than group II introns
Share a conserved secondary structure, which includes an “internal guide sequence” base-pairing with the 5’ splice site sequence in the upstream exon. The tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor

The similarity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterification

RNA Splicing Topic 6 RNA EDITING

RNA editing is another way of changing the sequence of an mRNA
I. Site specific deamination : 1. A specifically targeted C residue within mRNA is converted into U by the deaminase. 2. The process occurs only in certain tissues or cell types and in a regulated manner. RNA editing

The human apolipoprotein gene
Stop code In liver In intestines

3. Adenosine deamination also occurs in cells
3. Adenosine deamination also occurs in cells. The enzyme ADAR (adenosine deaminase acting on RNA) convert A into Inosine. Insone can base-pair with C, and this change can alter the sequence of the protein. 4. An ion channel expressed in mammalian brains is the target of Adenosine deamination.

II Guide RNA-directed uridine insertion or deletion.
1. This form of RNA editing is found in the mitochondria of trypanosomes. 2. Multiple Us are inserted into specific region of mRNAs after transcription (or US may be deleted).

3. The addition of Us to the message changes codons and reading frames, completely altering the “meaning” of the message. 4. Us are inserted into the message by guide RNAs (gRNAs) .

gRNAs Having three regions:
anchor– directing the gRNAs to the region of mRNAs it will edit. editing region – determining where the Us will be inserted poly-U stretch

RNA Splicing Topic 7 mRNA TRANSPORT

Once processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translation
mRNA transport All the fully processed mRNAs are transported to the cytoplasm for translation into proteins

Movement from the nucleus to the cytoplasm is an active and carefully regulated process.
The damaged, misprocessed and liberated introns are retained in the nucleus and degraded. A typical mature mRNA carries a collection of proteins that identifies it as being ready for transport. Export takes place through the nuclear pore complex.

Once in the cytoplasm, some proteins are discarded and are then imported back to the nucleus for another cycle of mRNA transport. Some proteins stay on the mRNA to facilitate translation.

Key points Why RNA splicing is important?
Chemical reaction: determination of the splice sites, the products, trans-splicing Spliceosome: splicing pathway and finding the splice sites Self-splicing introns and mechanisms Alternative splicing and regulation, alternative spliceosome Two different mechanisms of RNA editing mRNA transport-a link to translation

Section III: Expression of the Genome

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