Part II The Transfer of Genetic Information

Chapter 3 Transcription

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering College of Life Science and Technology, Xinjiang University

Li yi-jiejaylixj@hotmail.com

Content: Some basic conception Transcription in the Prokaryotic Nucleus Transcription in the Eukaryotic Nucleus mRNA Differences between Prokaryotes and

eukaryotes Production of mature mRNA in Eukaryotes Transcription in No Code Protein Genes

GeneA region of a DNA molecule containing a sequence of

bases that is transcribed into a functional product.

Several regions are responsible for the proper function of a gene.Regulatory region-sequence of bases that control the

initiation of transcription.

Coding region- sequence of bases that are read into a functional molecule (RNA or protein).

Termination region-sequence of nucleotides that stops transcription.

When a protein is needed by a cell, the genetic code for that protein must be read from the DNA and processed.

A two step process:

1. Transcription = synthesis of a single-stranded RNA molecule using the DNA template (1 strand of DNA is transcribed).

In Eukaryotes, transcription takes place in the nucleus.

2. Translation = conversion of a messenger RNA sequence into the amino acid sequence of a polypeptide (i.e., protein synthesis)

Translation takes place on ribosomes in the cytosol, or on rough ER

Both processes generally occur throughout the cell cycle.

Template strand: The 3’ to 5’ strand of DNA in a coding region that serves as the template for RNA synthesis. The resulting RNA is a complement of the template strand.

Nontemplate strand: The 5’ to 3’ strand of DNA in a coding region that has no function in producing the RNA. It has the same sequence as the initially transcribed RNA, except that Thymine in the DNA is Uracil in the RNA.

Sense/antisense +/- Nontranscribed/transcribed Nontemplate/template coding/template

Conventions for describing the strands

Both strands encode genes

Either strand of a DNA molecule may be used as a template, but transcription always reads the 3’ to 5’ strand relative to the direction of RNA polymerase movement.

mRNA--(messenger) intermediate molecules used for transfer of information from DNA to protein.

rRNA--(ribosomal) functional RNA molecules that are components of the ribosome.

tRNA--(transfer) functional RNA molecules that serve as adapters in translation.

snRNA--(small nuclear) functional RNA molecules that are involved in the removal of introns from pre-mRNAs.

scRNA--(small cytoplasmic) functional RNA molecules that are involved in protein traficking with the cytoplasm.

Various other functional RNAs

Five different types of RNA:

Unlike DNA replication, we will only want to copy specific sequences at specific times in specific cell types

We will need specific start and stop signals

Only one strand will be copied

How is an RNA strand synthesized?1. Regulated by gene regulatory elements within each gene.

2. DNA unwinds next to a gene.

3. RNA is transcribed 5’ to 3’ from the template (3’ to 5’).

4. Similar to DNA synthesis, except:

RNA polymerase

No primer

No proofreading

NTPs instead of dNTPs (no deoxy-)

Adds Uracil (U) instead of thymine (T)

Transcription in the Prokaryotic Nucleus

There is only one RNA polymerase in E. coli

Responsible for the synthesis of all types of RNA

We will first look at the core enzyme and then at the holoenzyme

Ds-DNA dependentdependent

E. coli RNA polymerase

Capable of polymerization but not initiation

RNA polymerase core enzyme

Core enzyme + sigma factor The sigma factor is a separate protein

required for initiation There are many different sigma factors Different sigma factors allow for the

expression of different genes

RNA polymerase holoenzyme

’ ’ + holoenzyme core polymerase sigma factor

The roles of each The roles of each subunitsubunit

The roles of each The roles of each subunitsubunit

Functions of other subunits:1. - binds the UP element found upstream of very strong

promoters (rRNA), and activators . - active site of Pol, also binds nascent RNA, RNA-

DNA hybrid, and DS DNA in front of the bubble 3. ’ – also binds nascent RNA, RNA-DNA hybrid, and DS

DNA in front of the bubble

4. -factor confers specificity to the polymerases, directs polymerase to initiate at specific sites, called promoters.

Phosphodiester bond formationDNA binding

DNA binding

Assemble holoenzyme

大肠杆菌中的 σ 因子能识别并与启动子区的特异性序列相结合

因子 基因 功能 -35 区 间隔（ bp ） -10 区

σ70 rpoD 广泛 TTGACA 16-18 TATAAT

σ32 rpoH 热休克 TCTCNCCCTTGAA 13-15 CCCCATNTA

σ54 rpoN 氮代谢 CTGGNA 6 TTGCA

Loose Loose

Looser Tightpromoter sites

non-promoter sites

non-promoter sites

promoter sites

Pol Core has non-specific affinity for DNA that is reduced by .

The function of sigma factor

• the sigma subunit of RNA polymerase is an “initiation factor”• there are several different sigma factors in E. coli that are

specific for different sets of genes• sigma factor functions to ensure that RNA polymerase binds

stably to DNA only at promoters• sigma destablizes nonspecific binding to non-promoter DNA• sigma stabilizes specific binding to promoter DNA• this accelerates the search for promoter DNA

Ka (M-1) Any DNA Promoter DNA(nonspecific) (specific)

Core 2 X 1011

Holo 1 X 107 1013 to 1015

• promoters vary in “strength” by ~two orders of magnitude

Core Enzyme 具有的四个功能位点

αα

β’

β

★ DNA/RNA 杂交位点 (β)

★ D. S.DNA 解链位点 (α) ★ D. S.DNA 重旋 (α) ★ 启动子识别位点 σ

★ DNA 无义链结合位点 (β’ )

The fidelity of RNA The fidelity of RNA polymerasepolymerase

The fidelity of RNA The fidelity of RNA polymerasepolymerase

No proofreading nuclease activities 1 error in 105 bp About 105 lower than replication

PromotersPromotersPromotersPromoters A DNA sequence that directs the

transcription of adjacent segments of the DNA

Consensus sequenceConsensus sequenceConsensus sequenceConsensus sequence

GATCT GATAT GACCT GTTCT AATCT Consensus: GATCT

Recognition sequences in the Recognition sequences in the promoter promoter

Recognition sequences in the Recognition sequences in the promoter promoter

The Primbnow box which includes -10 consensus sequence: TATAAT -35 consensus sequence: TTGACA

How does RNA polymerase find How does RNA polymerase find the promoter?the promoter?

How does RNA polymerase find How does RNA polymerase find the promoter?the promoter?

RNA polymerase scans DNA linearly Slides along DNA without opening strands Detects sequences of -10 and -35 in the major

groove Sigma factor allows RNA polymerase to bind

to promoter

The first events at the promoterThe first events at the promoterThe first events at the promoterThe first events at the promoter

RNA polymerase binds to the -35 region This is called a closed complex because the

DNA is still base-paired

Steps of Transcription

1. Banding

2. Initiation

3. Elongation

4. Termination

Occur in both prokaryotes and eukaryotes. Elongation is conserved in prokaryotes and eukaryotes. Initiation and termination proceed differently.

Step 2-Initiation --Transcription begins by the assembly of the RNA polymerase holoenzyme on a promoter region.

E. coli model:

Each gene has three regions:

1. 5’ Promoter, attracts RNA polymerase e.g., -10 bp 5’-TATAAT-3’e.g., -35 bp 5’-TTGACA-3’

2. Transcribed sequence, or RNA coding sequence3. 3’ Terminator, signals the stop point Minus numbers represent bases upstream of mRNA start point, +1 is the first base in

the RNA transcript.

1. RNA polymerase combines with sigma factor (a polypeptide) to create RNA polymerase holoenzyme

Recognizes promoters and initiates transcription.

Sigma factor required for efficient binding and transcription.

Different sigma factors recognize different promoter sequences.

2. RNA polymerase holoenzyme binds promoters and untwists DNA

e.g., binds loosely to the -35 promoter (DNA is d.s.)

e.g., binds tightly to the -10 promoter and untwists

3. Different types and levels of sigma factors influence the level and dynamics of gene expression (how much and efficiency).

Initiation

Polymerase bound tightly to promoter, “Closed Complex”

Polymerase unwinds DNA (-10 to +3), “Open Complex”

Starts synthesizing RNA

Step 3-Elongation… --RNA polymerization within a moving bubble (about 18 bases) of melted helix. Ribonucleoside triphosphates serve as the precursors

for synthesizing RNA. Except for the first, all ribonucleotides are added to an

existing 3’ hydroxyl group of an existing nucleotide.– Two external phosphates are lost as the internal forms a

phosphodiester bond with the hydroxyl group at the 3’ carbon of the ribose.

Due to this chemistry, an RNA molecule grows at its 3’ end, thus is synthesized in a 5’ to 3’ direction.

The sequence of the RNA is dictated by the template strand of DNA, which is organized 3’ to 5’ relative to the direction of transcription.

1. After 8-9 bp of RNA synthesis occurs, sigma factor is released and recycled for other reactions.

2. RNA polymerase completes the transcription at 30-50 bp/second.

3. DNA untwists rapidly, and re-anneals behind the enzyme.

4. Part of the new RNA strand is hybrid DNA-RNA, but most RNA is displaced as the helix reforms.

A. After ~10 nucleotides have been added, 5’ end ribonucleotide unpairs from template.

B. The subunit dissociates from core.

C. The size of RNA-DNA hybrid maintained during elongation.

D. Sigma recycles to new polymerase molecules.

Step 4-Termination… --stopping the synthesis of an RNA

Different mechanisms of termination

Prokaryotes– rho-independent termination: formation of a hairpin

structure

– rho-dependent termination: external protein disrupts transcription

Eukaryotes– cleavage of the RNA by an external protein

Two types of terminator sequences occur in prokaryotes:

1. Type I (-independent)

Palindromic, inverse repeat forms a hairpin loop and is believed to destabilize the DNA-RNA hybrid.

2. Type II (-dependent)

Involves factor proteins, believed to break the hydrogen bonds between the template DNA and RNA.

Rho Rho independentindependentRho Rho independentindependent Hairpins and poly(U) stretch Hairpins are formed by transcription of

palindromes

“Hairpin”

Weak associationA:U base pairs are weaker than G:C’s.

Stop signals exist at the end of the Stop signals exist at the end of the transcripttranscript

Stop signals exist at the end of the Stop signals exist at the end of the transcripttranscript

The terminated messageThe terminated messageThe terminated messageThe terminated message

Rho dependentRho dependentRho dependentRho dependent

Rho factor has an ATP-dependent RNA-DNA helicase activity

Still have a hairpin but no poly(U) stretch In both cases, the signal to terminate

transcription is present in the newly transcribed RNA, not in the DNA

Rho dependent terminationRho dependent terminationRho dependent terminationRho dependent termination

Rho-Dependent:

“Rho” is a bacterial Termination Factor

It acts as a hexamer of 46 kD subunits

- binds a specific 72 base sequence of ssRNA

It then hydrolyses ATP and eventually disrupts pairing between the nascent strand & template

Polymerase movement and supercoiling

Transcription in the Eukaryotic Nucleus

Prokaryotes possess only one type of RNA polymerase transcribes mRNAs, tRNAs, and rRNAs

Transcription is more complicated in eukaryotes

Eukaryotes possess three RNA polymerases:1. RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S

2. RNA polymerase II, transcribes mRNAs and some snRNAs

3. RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs

*S values of rRNAs refer to molecular size, as determined in a sucrose gradient (review box 5.1)

Polymerase Genes transcribed Subcellularlocalization

sensitivity to amanitin

RNApolymerase I

rRNA genesthis is actually the bulkof cellular transcription

nucleolus insensitive

RNApolymerase II

protein encoding genesare transcribed toproduce mRNA

nucleoplasm (everythingbut thenucleolus)

usually veryensitive

RNApolymeraseIII

tRNAs, 5S RNA and othersmall nuclear RNAs

nucleoplasm sensitivitydepends onspecies

Summary of RNAP roles and location

Promoters for the 3 nuclear Promoters for the 3 nuclear RNA polymerases (nRNAPs)RNA polymerases (nRNAPs)

Order of lecture topics:

1. Class II promoters (for nRNAP II)

2. Class I promoters (for RNAP I)

3. Class III promoters (for RNAP III)

4. Enhancers and Silencers

RNAP II Promoters (a.k.a. Class II) Class-II promoters usually have 4 components:

1. Upstream element2. TATA Box (at approx. –25)3. Initiation region

4. Downstream element

Many class II promoters lack 3 and 4.

1. 2. 3. 4.

TATA Box of Class II PromotersTATA Box of Class II Promoters

TATA box = TATAAAA Defines where transcription starts. Also required for efficient transcription for

some promoters. Some class II promoters (e.g., for

housekeeping genes or some developmentally regulated genes (e.g., homeotic)) don’t have a TATA box.

Transcription starts at a purine ~25-30 bp from the TATA box. Transcription starts at a purine ~25-30 bp from the TATA box.

SV40 early promoter analyzed in vivo.

Normal promoter.

Upstream Elements of Class IIUpstream Elements of Class II

Can be several of these Two that are found in many class II promoters:

1. GC boxes (GGGCGG and CCGCCCC)– Stimulate transcription in either orientation– May be multiple copies– Must be close to TATA box (different from

enhancers)– Bind the Sp1 factor

2. CCAAT box– Stimulates transcription– Binds CCAAT-binding transcription factor (CTF) or

CCAAT/enhancer-binding protein (C/EBP)

Class I Promoters (for nRNAP I)Class I Promoters (for nRNAP I)

Sequences less conserved than Class II Usually 2 parts:

– UCE : upstream control element , -150 to -100 in human rRNA

– Core: from - 45 to +20

Spacing between elements also important

-150 -100 -50 +20

UCE Core

Class III promoters (for nRNAP III)Class III promoters (for nRNAP III) 2 types:1. Internal promoters

- 5S rRNA (Box A, Intermediate element, Box C)- tRNA (Box A, B)

2. Class II – like promoters- contain TATA box- 7SL gene, promoter is upstream of coding region

Enhancers and SilencersEnhancers and Silencers

Enhancers stimulate transcription, silencers inhibit.

Both are orientation independent.– Flip 180 degrees, no effect

Both are position independent.– Can work at a distance from promoter– Enhancers have been found all over

Bind regulated transcription factors.

Transcription Factors for Class II Transcription Factors for Class II promoters (RNAP II)promoters (RNAP II)

• Basal factorsRequired for initiation at nearly all promoters; determine site of initiation; interact with TATA box.

• Upstream factorsDNA binding proteins that recognize consensus elements upstream of TATA box. Ubiquitous. Increase efficiency of initiation. Interact with proximal promoter elements (e.g., CCAAT box).

• Inducible (regulated) factorsWork like upstream factors but are regulatory. Made or active only at specific times or in specific tissues. Interact with enhancers or silencers.

Transcription of protein-coding genes by RNA polymerase II

Basal transcription factors (TFs) also are required by RNA polymerases.

TFs are proteins, assembled on basal promoter elements

Each TF works with only one kind of RNA polymerase (required by all 3 RNA polymerases).

Numbered (i.e., named) to match their RNA polymerase.

e.g., TFIID, TFIIB, TFIIF, TFIIE, TFIIH

Binding of TFs and RNA polymerase occurs in a set order in protein coding genes.

Complete complex (RNA polymerase + TFs) is called a pre-initiation complex (PIC).

Order of binding is: IID + IIB + RNA poly. II + IIF +IIE +IIH

Binding of Activator Factors

Finally, high-level transcription is induced by the binding of activator factors to DNA sequences called enhancers.

Single or multiple copies in either orientation

Usually located upstream

Can be several kb from the gene

Silencer elements and repressor factors also exist

mRNA Differences between Prokaryotes and eukaryotes:

Prokaryotes

1. mRNA transcript is mature, and used directly for translation without modification.

2. Since prokaryotes lack a nucleus, mRNA also is translated on ribosomes before is is transcribed completely (i.e., transcription and translation are coupled).

3. Prokaryote mRNAs are polycistronic, they contain amino acid coding information for more than one gene.

Eukaryotes

1. mRNA transcript is not mature (pre-mRNA) and must be modified by processing.

2. Transcription and translation are not coupled (mRNA must first be exported to the cytoplasm before translation occurs).

3. Eukaryote mRNAs are monocistronic, they contain amino acid for just one gene.

Prokaryotes and Eukaryotes

Production of mature mRNA in eukaryotes

Multiple Steps in Gene Expression after Transcription

1. Transcription

2. 5’ Capping

3. 3' maturation: cleavage & polyadenylation

4. Splicing

5. Editing

6. Transport of RNA to Cytoplasm

7. Stabilization/Destabilization of mRNA

8. Translation

Structure of a eukaryotic gene

1. Gene is transcribed by RNA polymerase II.

2. Introns are removed and exons are spliced.

Post-transcriptional processing of mRNA in eukaryotes

Initial transcript is known as the pre-mRNA, and before being exported from the nucleus processing must occur. Final processed mRNA is exported to the cytoplasm.

RNA processing:

1. Addition of 7-methylguanosine cap to the 5’ end by guanyltransferase.

2. Addition of poly-Adenosine tail at the 3’ end by poly(A) polymerase.

3. Removal of introns by the spliceosome

RNA Capping

1. Post-transcriptional (i.e., G not encoded)2. Involves adding a 7MeGuanosine Nt to the first (RNA) Nt in

an unusual way, and often methylation of the first few nt of the RNA.

3. Occurs before the pre-mRNA is 30 nt long

4. The 5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotide.

– Results in the addition of two methyl (CH3) groups.

– Essential for the ribosome to bind to the 5’ end of the mRNA.

AdoMet = S-adenosylmethionine, the methyl donor

Capping: order of events and enzymes

Product is Cap 1

or “RNA triphosphatase”

Cap Functions

Cap provides:

1. Protection from some ribonucleases

2. Enhanced translation

3. Enhanced transport from nucleus

4. Enhances splicing of first intron for some mRNAs

Post-transcriptional Processes II: Pre-mRNA Polyadenylation

Most cytoplasmic mRNAs have a polyA tail (3’ end) of 50-250 Adenylates– a notable exception is histone mRNAs

Added post-transcriptionally by an enzyme, polyA polymerase(s)

Turns over (recycles) in cytoplasm

Functions of the PolyA Tail1. Promotes mRNA stability

- Deadenylation (shortening of the polyA tail) can trigger rapid degradation of the mRNA

2. Enhances translation

- promotes recruitment by ribosomes

- bound by a polyA-binding protein in the cytoplasm called PAB1

- synergistic stimulation with Cap!

Methods: Firefly luciferase mRNAs (with 5’ and 3’ UTRs from a plant gene) were electroporated into protoplasts. At intervals, the amount of luciferase mRNA was checked (to determine half-life), and the amount of luciferase activity (which reflects the amount of translation product).

Overview of Polyadenylation Mechanism

1. Transcription extends beyond mRNA end

2. Transcript is cut at 3’ end of what will become the mRNA (in green)

3. PolyA Polymerase adds ~250 As to 3’ end

4. “Extra” RNA (in red) degraded

Evidence for Transcription Past the End of a Cellular mRNA

Nuclear run-on transcription assay using Friend erythroleukemic cells treated with DMSO (stimulates globin gene transcription)– Newly synthesized RNAs are hybridized to DNA

regions that span the globin gene, including regions downstream

– The amount of newly synthesized RNA complementary to each gene region is thus quantified

Run-on transcription assay with isolated nuclei

Fig. 5.33 Only gene Y is transcribed.

Transcription beyond the polyA site.

After run-on transcription (in the presence of 32P-UTP) with nuclei from Friend Erythroleukemic cells, the labeled RNA was hybridized to DNA fragments A-F that span the globin gene. The relative molarities of newly synthesized RNA that hybridized to each fragment are given. s.d. is the standard deviation.

Notice that there is just as much RNA transcribed from downstream of the gene (E) as there is within the gene (C).

Polyadenylation (PolyA) Signals AAUAAA in mammals and plants Located ~20-30 bp from the polyA site

– Other hexamers less efficient but are used Mutagenesis and in vivo expression studies

reveal 2 other motifs downstream of AAUAAA that are important:

1. GU-rich stretch2. U-rich stretch

What if there are multiple possible signals?

Competition experiment:1. The synthetic polyadenylation site (SPA) below

was inserted downstream of a normal one in a globin gene

Fig. 15.21

GU-rich U-rich

2. The modified globin gene was introduced into HeLa cells, and the 3’ end of the mRNA analyzed by S1 mapping

Result: SPA was mainly used for polyAdenylation

Conclusion Stronger set of signals in the SPA, which

“outcompeted” the native globin polyA signal (it lacks the U-rich motif)

Polyadenylation: The ProteinsSeveral proteins are required in mammals for cleavage and

polyadenylation.

Proteins required for efficient cleavage of pre-mRNA:1. CPSF (cleavage and polyadenylation specificity

factor), binds the AAAUAA2. CstF (cleavage stimulation factor) binds to the G/U

rich region cooperatively with CPSF3. CFI and CFII (cleavage factors I and II), RNA-binding

proteins4. PAP (poly A polymerase) 5. nRNAP II (the CTD of the very large RPB1 subunit)

stimulates cleavage.

Model for the pre-cleavage complex.

Fig. 15.26

Polyadenylation: Mechanism Occurs in 2 phases

– Phase 1: requires AAUAAA and ~8 nt downstream (3’)

– Phase 2 : Once ~10 As are added, further adenylation does not require the AAUAAA

2 Phases to Polyadenylation

Fig. 15.28

substrates:1. 58-nt RNA from SV40 that ends with AAUAAA plus 8 nt2. same RNA as in 1 plus A40 tail3. same RNA as in 1 plus a 40-nt 3’ tag from a vector sequence

The series with an X contain a mutated AAUAAA (AAGAAA).

Hela cell nuclear extract incubated with various radiolabeled RNA substrates, which were then separated by gel electrophoresis.

Conclusion: the AAUAAA not needed for phase 2, only phase 1.

Proteins Required for Polyadenylation

Phase I:1. CPSF 2. PolyA polymerase

Phase II:1. PolyA polymerase2. PolyA Binding Protein II (PAB II)

- PAB II binds to short A-tail- Helps PAP synthesize long tails

Nuclear PolyA Polymerase (long form = PAPII)

RBD - RNA binding domainNLS - nuclear localization signalPM - polymerase moduleS/T- serine/threonine rich regions (yellow)

CPSF specificity factor

CFI and CFII

PAP II

CstF stimulation factor

PAB II

Fig. 15.32

RNA Splicing

RNA splicing is the removal of intervening sequences (IVS) that interrupt the coding region of a gene

Excision of the IVS (intron) is accompanied by the precise ligation of the coding regions (exons)

Introns and exons:

Eukaryote pre-mRNAs often have intervening introns that must be removed during RNA processing (as do some viruses).

intron = non-coding DNA sequences between exons in a gene.

exon = expressed DNA sequences in a gene, code for amino acids.

1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)

mRNA splicing of exons and removal of introns:

1. Introns typically begin with a 5’-GT(U) and end with AG-3’.

2. Cleavage occurs first at the 5’ end of intron 1 (between 2 exons).

3. The now free G joins with an A at a specific branch point sequence in the middle of the intron, using a 2’ to 5’ phosphodiester bond.

Intron forms a lariat-shaped structure.

4. Lariat is excised, and the exons are joined to form a spliced mRNA.

5. Splicing is mediated by splicosomes, complexes of small nuclear RNAs (snRNAs) and proteins, that cleave the intron at the 3’ end and join the exons.

6. Introns are degraded by the cell.

Intron Classes & Distribution

1. Group I - common in organelles, nuclear rRNA genes of lower eukaryotes, a few prokaryotes and some phage genes

2. Group II - common in organelles, also in some prokaryotes

3. Nuclear mRNA (NmRNA) - ubiquitous in eucaryotes

4. Nuclear tRNA- some eucaryotes

Relationships of the 4 Intron

Classes 1. Each class has a distinctive structure

2. The chemistry of the Groups I, II and NmRNA reactions are similar – i.e, transesterification reactions

3. The splicing pathway for Group II and nuclear mRNA introns are similar

4. Splicing of Groups I, II and possibly NmRNA introns are RNA-catalyzed

Self-Splicing Introns

1. Some Group I and Group II introns can self-splice in vitro in the absence of proteins (or other RNAs), i.e. they are ribozymes.

2. Each group has a distinctive, semi-conserved secondary structure.

3. Both groups require Mg2+ to fold into a catalytically active ribozyme.

4. Group I introns also require a guanosine nucleotide in the first step.

Almost all introns begin with “GU”and end with “AG”

1. A snurp contains a small, nuclear U-rich RNA (snRNAs = U1, U2, U4, U5 or U6), and proteins (at least 7).

2. The snRNAs base-pair with the pre-mRNA (U1, U2, U5, U6), and some with each other (U4-U6 pair in snurps, and U2-U6 pair in spliceosome).

3. Lupus patients have antibodies to snurps.

Spliceosomes contain Snurps (a.k.a., snRNPs or small nuclear

ribonucleoprotein particles)

1. U1 base-pairs with the 5’ splice-site2. U2 binds/pairs with the branch point; also pairs

with U6 in the assembled spliceosome3. U4 pairs with U6 in SnRNPs, but unpairs during

spliceosome assembly 4. U5 interacts with both exons (only 1-2 nt adjacent to

intron); helps bring exons together 5. U6 displaces U1 at the 5’ splice-site (pairs with nt in

the intron); it also pairs with U2 in the catalytic center of the spliceosome

Roles of snRNAs/Snurps

U1 and U2 paired with pre-mRNA in yeast

Fig. 14.26

Similar active sites (catalytic center) in Spliceosomal and Group II introns?

(both models after first step)

The yeast Spliceosome cycle of assembly, Rxn, and disassembly

Fig. 14.29

RNA Editing Definition: any process, other than splicing,

that results in a change in the sequence of a RNA transcript such that it differs from the sequence of the DNA template

1. Adds or deletes nucleotides from a pre-RNA, or chemically alters the bases, so the mRNA bases do not match the DNA sequence.

2. Results in the substitution, addition, or deletion of amino acids (relative to the DNA template).

3. Generally cell or tissue specific.

Examples: protozoa, slime molds, plant organelles, mammals

Discovered in trypanosome mitochondria Also common in plant mitochondria Also occurs in a few chloroplast genes of

higher plants, and at least a few nuclear genes in mammals

Discovery of RNA Editing in Trypanosome Mitochondria

Unusual Mitos. called Kinetoplasts DNA:

– Maxicircles (22 kb in T. brucei), contains most of the genes

– Minicircles (1-3 kb), heterogenous Sequencing of genomic Mt DNA (Maxicircles)

revealed apparent pseudogenes:– Full of Stop codons– Deletions of important amino acids

Kinetoplast DNA from a trypanosome visualized by EM

Where were the real functional genes?

Investigators generated cDNA clones to some of the kinetoplast mRNAs and sequenced them

Sequences were partially complementary to pseudogenes on maxicircle DNA

e.g., cytochrome oxidase subunit III

– the COXIII DNA sequence above is missing 4 Us found in the mRNA

Called this “Editing” because it produced functional mRNAs and proteins from pseudogenes

COXIIICytochrome oxidase III

From Trypanosoma brucei

Lower case Us were inserted by editing.The deleted Ts (found in the DNA) are indicated in upper case.

Some genes are very heavily edited!

Editing Mechanism Post-transcriptional Guide RNAs (gRNAs) direct editing

– gRNAs are small and complementary to portions of the edited mRNA

– Base-pairing of gRNA with unedited RNA gives mismatches, which are recognized by the editing machinery

– Machinery includes an endonuclease, a Terminal UridylylTransferase (TUTase), and a RNA ligase

Editing is directional, from 3’ to 5’

Guide RNAs Direct Editing in Trypanosomes.

Editing is from 3’ to 5’ along an unedited RNA.

TUTase: Terminal Uridylyl Transferase

Editing Mechanism with the enzymes.

Other Systems with RNA Editing Land plant (C U) and Physarum (slime

mold) mitochondria (nt insertions) Chloroplasts of angiosperms (C U) A few nuclear genes in mammals

– Apolipoprotein B (C U)– Glutamate receptor [A I (inosine)]

Hepatitus delta virus (A I) Paramyxovirus (G insertions)

Post-Transcriptional Gene Silencing (PTGS)

Also called RNA interference or RNAi. Process results in down-regulation of a

gene at the RNA level (i.e., after transcription).

There is also gene silencing at the transcriptional level (TGS).

Examples: transposons, retroviral genes, heterochromatin

Discovery of PTGS First discovered in plants (1990) Introduction of transgenes homologous to

endogenous genes often resulted in plants with both genes suppressed!

Called Co-suppression Resulted in degradation of the endogenous

and the transgene mRNA

Discovery of PTGS (cont.) Involved attempts to manipulate pigment synthesis

genes in petunia Genes were enzymes of the flavonoid/

anthocyanins pathway: – CHS - chalcone synthase– DFR - dihydroflavonol reductase

When these genes were introduced into petunia using a strong viral promoter, mRNA levels dropped and so did pigment levels in many transgenics

Flavonoid/anthocyanin pathway in plants

Strongly pigmented compounds

DFR construct introduced into petuniaCaMV - 35S promoter from Cauliflower Mosaic VirusDFR cDNA – cDNA copy of the DFR mRNA (intronless DFR gene)T Nos - 3’ processing signal from the Nopaline synthase gene

Flowers from 3 different Transgenic petunia plants that carry copies of the chimeric DFR gene above. These flowers showed reduced DFR mRNA levels (lower than wild-type) in the non-pigmented areas.

Antisense Technology Antisense technology has been used for ~20

years Based on introducing an antisense gene (or

antisense RNA) into cells or organisms to try to block translation of the sense mRNA.

Alternative to gene knock-outs, which are very difficult to do in higher plants and animals.

The “antisense effect” was probably due to RNAi rather than inhibiting translation

RNAi discovered in C. elegans (the first animal) while attempting to use antisense RNA in vivo

– “Sense” control RNAs also produced suppression of target gene!

– turned out that the sense (and antisense) RNAs were contaminated by small amounts of dsRNA

– dsRNA was the suppressing agent

Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode C. elegans.

Antisense RNA (c) or dsRNA (d) for the mex-3 (mRNA) was injected into C. elegans ovaries, and then mex-3 mRNA was detected in embryos by in situ hybridization with a mex-3 probe.(a) control embryo(b) control embryo hyb. with mex-3 probe

Conclusion: dsRNA reduced mex-3 mRNA better than antisense mRNA. Also, the suppression signal moves from cell to cell.

Fig. 16.37

PTGS occurs in wide variety of in Eukaryotes

called RNA interference or RNAi in:– C. elegans (nematode)– Drosophila– Mammalian cells

called “quelling” in Neurospora

Not detected (yet) in Yeast!

Mechanism of RNAiSome facts and findings:1. Cells (plants and animals) undergoing RNAi contain small

RNAs (~25 nt) that seem to result from degradation of the target mRNA.

2. A nuclease was purified from Drosophila embryos undergoing RNAi that digested the target mRNA.

• The nuclease contained associated small RNAs (both sense and antisense)

• Degradation of the small RNAs with micrococcal nuclease prevented the RNAi nuclease from degrading the target mRNA

3. Facts suggest that a nuclease digests dsRNA into small fragments, which initiate the RNAi process by activating and guiding the nuclease to the mRNA.

Generation of 21-23 nt fragments of RNA in a RNAi-competent Drosophila embryo extract.

dsRNA of luciferase reporter genes were added to the reaction (lanes 2-10), in the presence or absence of the corresponding mRNA. The dsRNAs were labeled on the sense, antisense or both strands. Lanes 11, 12 contained 32P-labeled, capped antisense Rluc RNA.

Fig. 16.38

The dsRNA that is added dictates where the destabilized mRNA is cleaved.

The dsRNAs A, B, or C were added to the Drosophila extract together with a Rr-luc mRNA that is 32P-labeled at the 5’ end. The RNA was then analyzed on a polyacrylamide gel and autoradiographed.

Results: the products of Rr-luc mRNA degradation triggered by RNA B are ~100nt longer than those triggered by RNA C (and ~100 nt longer again for RNA A induced degradation).

Fig. 16.39

High resolution gel analysis of the products of Rr-luc mRNA degradation from the previous slide.

Results: the cleavages occur mainly at 21-23 nt intervals. There is an exceptional cleavage only 9 nt from an adjacent cleavage for RNA C. This cleavage occurred at a stretch of 7 Us. 14 of 16 cleavage sites were at a U.

Model for RNAiBy “Dicer”

21-23 nt RNAs

Fig. 16.41

ATP-dependentHelicase?

Active siRNA complexes.

Very efficient process because many small interfering RNAs (siRNAs) generated from a larger dsRNA.

Biological Significance of RNAi

Most widely held view is that RNAi evolved to protect the genome from viruses (or other invading DNAs or RNAs)

Recently, very small (micro) RNAs have been discovered in several eukaryotes that regulate developmentally other large RNAs– May be a new use for the RNAi mechanism besides

defense

Transcription in No code protein Genes

1. rRNA, ribosomal RNA

Catalyze protein synthesis by facilitating the binding of tRNA (and their amino acids) to mRNA.

2. tRNA, transfer RNA

Transport amino acids to mRNA for translation.

3. snRNA, small nuclear RNA

Combine with proteins to form complexes used in RNA processing (e.g., the splicosome).

1. Synthesis of ribosomal RNA and ribosomes:

1. Cells contain thousands of ribosomes.

2. Consist of two subunits (large and small) in prokaryotes and eukaryotes, in combination with ribosomal proteins.

3. E. coli 70S model:

50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins

30S subunit = 16S (1,542 nt) + 20 proteins

4. Mammalian 80S model:

60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50 proteins

40S subunit = 18S (1,900 nt) + 35 proteins

5. DNA regions that code for rRNA are called ribosomal DNA (rDNA).

6. Eukaryotes generally have many copies of rRNA genes tandemly repeated.

1. Synthesis of ribosomal RNA and ribosomes:(continued):

7. Transcription occurs by the same mechanism as protein-coding genes, but generally using RNA polymerase I.

8. rRNA synthesis requires its own array transcription factors (TFs)

9. Coding sequences for RNA subunits within rDNA genes contain internal (ITS), external (ETS), and nontranscribed spacers (NTS).

10. ITS units separate the RNA subunits through the pre-rRNA stage, whereupon ITS & ETS are cleaved out and rRNAs are assembled.

11. Subunits of mature ribosomes are bonded together by H-bonds.

12. Finally, transported to the cytoplasm to initiate protein synthesis.

Mammalian example of 80S rRNA

2. Synthesis of tRNA:

1. tRNA genes also occur in repeated copies throughout the genome, and may contain introns.

2. Each tRNA (75-90 nt in length) has a different sequence that binds a different amino acid.

3. Many tRNAs undergo extensive post-transcription modification, especially those in the mitochondria and chloroplast.

4. tRNAs form clover-leaf structures, with complementary base-pairing between regions to form four stems and loops.

5. Loop #2 contains the anti-codon, which recognizes mRNA codons during translation.

6. Same general mechanism using RNA polymerase III, promoters, unique TFs, plus posttranscriptional modification from pre-tRNA.

3. Synthesis of snRNA (small nuclear RNA):

• Form complexes with proteins used in eukaryotic RNA processing, e.g., splicing of mRNA after introns are removed.

• Transcribed using RNA polymerase II or III.

Reference: CAIs: 1. Principles of Genetics: Kevin G. McCracken

– http://mercury.bio.uaf.edu/~kevin_mccracken/genetics/

2.Molecular Biology : Profs. Ding Xue and Ravinder Singh– http://mcdb.colorado.edu/labs/xue/

3. Molecular Biology : David L. Herrin, Ph.D. – http://www.esb.utexas.edu/herrin/bio344/

4. Dr. Eric Aamodt– http://www.sh.lsuhsc.edu/new_curric/mod1_1.html

5.Molecular Biology: 5.Molecular Biology: zhenyonglianzhenyonglian

– www.hzau.edu.cn

Books: 1.Molecular Biology (Third): Robet F.Weaver

– McGraw-Hill Companies, Inc. 2004

2.Genes VIII: Benjamin Lewin – Prentice Hall 2004

3. 现代分子生物学（第二版） 朱玉贤、李毅 – 高等教育出版社， 1997.3 ，

4. 分子生物学 阎隆飞 张玉麟，– 中国农业大学出版社， 1997.8 ，

5. 分子遗传学 孙乃恩主编 – 南京大学出版社， 1996 ，