Expression of Genome

Chapter 13 Mechanism of TranscriptionRNA polymerase and the transcription cycleThe transcription cycle in bacteriaTranscription in eukaryotesTranscription by RNA polymerases

Chapter 14 RNA Splicing

Chapter 15 Translation

Chapter 16 The Genetic Code

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Overview of RNA•Transcription: Synthesized of RNA using DNA template

•RNA are mainly single stranded, thus– Much greater structural diversity, suited a variety functions

•Most eukaryotic RNA are processed after synthesis– introns; poly-adenylation; capping

•Three main roles of RNA in relay of genetic information– messenger (mRNAs); transfer (tRNAs); translation (rRNAs)

•RNA also play less-understood functions – miRNA, snoRNA, snRNA, gRNA, ncRNA, lincRNA (long itergenic

noncoding), piRNA, …

•RNA, the only macromolecule play roles in storage (as genomic material in some viruses), in transmission of information and in catalysis (ribozyme), and the role in prebiotic life.

•Transcriptome: the sum of all the RNA produced in a cell under a given set of conditions. 2

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Nomenclature: Ribonucleotides

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Contrary to DNA, RNA is unstable

1. RNA is unstable under alkaline conditions

2. The 2’, 3’-vicinal diol of ribose is prone to be cleaved

by mild oxidizing agents

3. Hydrolysis is also catalyzed by enzymes (RNases)– S-RNase in plants prevents inbreeding– RNase P, ribozyme (RNA enzyme) that processes tRNA

precursors– Dicer, enzyme that cleaves ds-RNA into oligonucleotides

•protection from viral genomes•RNA interference technology

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Base-catalyzed RNA Hydrolysis

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Replication vs. Transcription

• Resembles:–Both initiation, elongation, and termination stages. –Both use ssDNA as template–Same polarity (direction of synthesis 5’ 3’)–Same mechanism: both add nucleotides via attack of 3’ -OH of

growing chain to -Pi of NTPs.

• Differs:–NTP not dNTP for transcription, pairs A＝U.–Primer, RNA pol initiates de novo–Fidelity, lower for txn, no catastrophic outcome , 1/104~5 for RNA;

DNA replication 1/1011 (with repair system)–Whole genome replicated; but only certain segments of DNA are

transcribed, or only a particular time gene transcribed, –Much of the interest in transcription regulation, largely initiation–Within each DNA segments, only one DNA strand as a template.–RNA product not base-paired to template DNA

DNA polymerases and RNA polymerases

There are other difference points:

Kinetic features, DNA pol III, ~1000 nt/sec, much higher than transcription—RNA pol, ~50 nt/sec (coupled trxn/trln), thus, trxnmuch slower but occurs at many sites, far more RNA in cells.

Processivity forDNA pol is low, but RNA pol has high processivity(# nt added before release) a must, as if trxn terminatedprematurely, have to start all over again.

Fidelity, lack a separate proofreading 3’5’ exonuclease. Though mistake in RNAs is less consequence to cells. RNA polymerases (both bacterial and eukaryotic), do pause when a mispaired base added.

In E coli, GreA/GreB promote 3’-hydrolytic cleavage of wrong base added direct reversal of the polymerase reaction. But usually removes dinucleotides, and reaction is slower.

Encounter certain DNA sequences results in a pause in RNA synthesis, and at some sequences trxn is terminated.

Template and nontemplate (coding) DNA strands

p.1024

• The two DNA strands have different roles in transcription.

• The strand as template is called the template strand.

• The complementary nontemplate (or coding) strand, is sequence identical to the transcribed RNA, with U place T.

• Both DNA strands may encode RNA products

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Ask:1. How the DNA double helix is unwound?2. How an RNA strand is built on the template strand? 3. How the RNA transcript dissociates from the DNA

template? And where to dissociate?4. how the DNA strands re-anneal?5. What factors or enzymes are involved and the

mechanism?

Transcription of DNA into RNA

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Discovery DNA polymerase I spurred hot search for RNA Polymerase.

•1st, polynucleotide phosphorylase, an RNA synthesing enzyme, quite different: n rNDP (rNMP)n + n Pi

But, it is unlikely, (no template req, random RNA seq)Also, no eukaryotic counterpart? shown later, not for RNA syn, but in degradation bacterial mRNAs. Of Great value, syn polynucleotides in cracking genetic codes.

By 1961, 4 gps independently detected, form RNA from rNTP (ribonucleoside 5’-triphosphates). Subseq, purified E coli RNA polymerase defines fundamental properties of transcription.

DNA-dependent RNA polymerase, requires DNA template, all four rNTPs (ATP, GTP, UTP, CTP), and Mg2+ (protein also binds one Zn2+).

elongates by adding NMP to 3’-OH end, building in 5’3’, 3’-OH as nucleophile, attacking -phosphate of incoming NTP and releasing PPi.

The overall reaction: (rNMP)n + rNTP (rNMP)n+1 + PPiRNA Lengthened RNA

DNA-Dependent Synthesis of RNA

5 4 3 2 1

RNA polymerase core and

holoenzyme

SDS-PAGE

1. Holoenzyme

2. Peak A

3. Peak B

4. Peak C

5. Purified

factor

12Phosphocellulose fractionation:

A, B, and C.

• E. coli RNA pol : complex with 5 core subunits (α2ββ‘ω, Mr 390 kD) and a 6th subunit.

• The is a group proteins (variants size). subunit binds transiently to the core and directs to specific sites on the DNA.

• Core enzyme: basic RNA pol activity but no specificity. (only ~30% w/o factor), adding factor restores specificity.

• Thus, factor for Specificityfactor, thus constitute the RNA polymerase holoenzyme.

Bacterial RNA Polymerase has Six Subunits

• Two subunits: assembly and binding to UP elements

• The subunit: main catalytic subunit

• The ’ subunit: responsible for DNA-binding

• The subunit: directs enzyme to the promoter

• The appears to protect the polymerase from denaturation 13

RNA Polymerases in Different Forms, but Share Features

Homology

1. two large subunits, ‘ and = RPB1 and RPB2.

2. The subunits = RPB3 and RPB11

3. The = RPB6.14

Crystal structures of prokaryotic and eukaryotic RNA polymerases.a) RNA pol core, T. aquaticus.

subunits: (blue) ; (purple) ’; (yellow/green) two sub-units; (red) . The Mg2+ ion (red ball), marks the active site in .

b) RNA Pol II of yeast S. cerevisiae. As to the bacterial enzyme. RPB1 and RPB2 in purple/blue; RPB3 and RPB11 in green/yellow; and RPB6 in red.

Mg2+

A crab claw, hand structure, pincer (active center cleft)

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Mg2+

’

RPB1

RPB2

RPB6

RPB3

RPB11

Catalytic mechanism of RNA Polymerase

• Mg++ on the right coordinates to the -phosphate to facilitate 3’-hydroxyl attack

• Mg++ on the left facilitates the displacement of PPi.

• Both Mg++ stabilizes the negatively charged transition state16

The phases of transcription: Initiation, elongation, and termination.

• The transcription start site designated the "+1" position.

• Sequences in downstream of *1 allotted “+” values. Those of upstream given a “−” value.

1. In initiation (3 steps)a)initial binding to promoter to

form the closed complex, b)melting of DNA to form the

open complex, c) initial transcription complex,

2. Elongation3. termination

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Transcription by RNA polymerase in E. coli

•RNA polymerase most active when DNA-bounded, covers ~40-bp DNA.

•Copied in 3’5’ direction•DNA transiently unwound– ~17 bp

(transcription bubble) ahead and rewound behind.

•Growing RNA pairs template ~8 bp•As DNA rewound, the ssRNA strand

extruded. • RNA polymerase action create “+”

supercoils (overwound) ahead, “-” supercoils (underwound) behind the bubble.

• Topoisomerases required.• Elongation: E coli RNA pol, ~ 50 - 90

nt/sec. Eukaryotic RNA pol: ~ 40 nt/sec.

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Three Defined Steps in Transcription Initiation

1. Forming a closed complex (bound one face of ds-DNA helix)

2. closed complex transition to the open complex.

DNA partially unwound over ~14 bp (-10 to +2/+ 3), form transcription bubble (AT-rich promoter region separated).

3. Form initial transcribing complex: first ~10 nts is inefficient, enzyme often releases short transcripts (< 10 nt).

4. Form stable ternary complex: once > ~ 10 bp escaped promoter. Transition to elongation phase. σ subunit dissociates.

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bacterial promoter by σ70 revealed two sequences at about –10 (5’-TATAAT-3’); –35 (5’-TTGACA-3’) separated by ~17 bp spacing.

A third AT-rich element: UP element, between -40/-60 in some promoters, as rRNA promoter.

UP element promotes strand separation during initiation.

The consensus elements in E coli promoters

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RNA polymerase footprinting on a promoter

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6.3 Transcription Initiation

The role of -subunit in

UP element recognition

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The “extended -10” and “discriminator” elements in E coli Some 70-promoters "extended -10" element, provide extra

contacts to compensate for the lack of -35 region. eg, the gal promoter of E. coli.

The strength of the interaction between discriminator and polymerase influences the stability of the enzyme-DNA complex.

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The -70 factor four regions (1 - 4)-10 and -35 elements by region 2 and 4, respectively.Region 2.3 for melting DNA. The extended-10 element by an α helix in region 3, which makes

contact with two specific bp in the element. The discriminator by region 1.2. Same distance: 75Å (-10 to -35) and -region (2 to 4)Region 1.1, molecular mimetic of DNA upon shifting close open.

The Factor Recognizes the Promoter

75 Å

75 Å

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The role of Region 4:

Region 4 form a DNA-binding motif: the α helix-turn-helix. A helix inserts into major groove, interacts w/ the -35 bases;

provides binding energy to secure polymerase to promoterthe other lies across the top of the groove, making contacts

with the DNA backbone.

Similar structural motif in many DNA-binding proteins, almost all transcriptional activators/repressors in bacterial.

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The and α subunits recruit RNA polymerase core to

promoter. The regions 2 and 4 recognize the ~10 and -35 regions, separated by 75A, same dist

2- 4 and ~10 and -35

the UP-element is not recognized by but is recognized by

the αCTD of α subunit.

The αCTD is connected to the αNTD by a flexible linker.

75 Å

75 Å

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The role of region 2 The -10 region also recognized by an α helix: has a more

elaborate role in DNA melting initiated the transition from the closed to open complex.

Not just for binding DNA: the α helix has several essential aromatic amino acids, that interact with bases on the nontemplate strand.

That stabilizes the melted DNA. Similar role for the single-strand binding protein (SSB) during DNA replication.

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Recognition and melting of the -10 element by region 2The energetically preferred bindings drive the melting of the promoter -10

The flipped out bases, A and T, drives melting.

The isomerization process (close open), not require ATP but a spontaneous conformational change in DNA-enzyme complex to a energetically favorable form. 28

Aromatic residue stacking with nitrogen

base in DNA or RNA polymerases

Overlapping of

aromatic pi-electron

clouds

This figure is DNA polymerase 29

Transition to the Open Complex

Formation of the closed complex, is readily reversible;

Transition from closed to open complex involves structural changes in the enzyme and the opening of the DNA double helix, "melting" occurs between positions

-11 and +3.

For 70, the transition called isomerization, does not require energy derived from ATP hydrolysis, is a spontaneous conformational change in the DNA-

enzyme complex to a more energetically favorable form.

Isomerization is essentially irreversible, guarantees that transcription will subsequently initiate.

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Locating and demonstrating the melting of DNA

promoter by RNA polymerase

Methylation of melted base-A by dimethyl sulfate (DMS), which

prevents reanneal when RNA pol is removed, is sensitive to S1

nuclease (ssDNA) 31

• RNA polymerase binding/methylation

with DMS (dimethyl sulfate to modify

the base, thus prevents reanneal), S1

nuclease digestion

• the melted region in T7 A3-promoter

RNA polymerase melts: -9 ~ +3 region

Locating and demonstrating the melting of DNA

promoter by RNA polymerase

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R+S+ = DMS-RNA and then S1 nuclease treated

GA: partial sequence for G/A.

Five channels into/out of the open complex.

• The active site, β + β‘, at base of pincers w/i "active center cleft.”

1. The RNA-exit channel

2. The downstream dsDNA channel (DNA enters, ds separate at +3).

3. The nontemplate-strand (NT) channel.

4. The template-strand (T) channel (double helix reforms at -11).

5. The nucleotide entry (NTP-uptake) channel, NTP to about “+1".

• region 3/4 linker (or 3.2) links 3.1 and 4.

• region 1.1, a DNA molecular mimetic (highly “-” charges), shift 50 A to outside of RNA pol, leaves space for DNA upon closeopen isomerization

• Space in RNA pol active centerhighly “+” charges (ie, fit -1.1 or DNA). 33

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Binding of -subunit to promoter and transcriptional activation by a single activator (via -subunit)

Transcription without the Need for a Primer

•RNA pol works without a primer• impressive feat requires DNA template brought into polymerase

active site and held stably in a helical conformation, and initiating 1st NTP be brought into active site and held stably on template while the 2nd NTP is presented. ie, 1st NTP set to correct geometry for polymerization

•particularly difficult because RNA pol starts most transcripts with an A, which with only two H-bonds.

•This requirement explains why most transcripts start with the same nucleotide. The interactions are specific for A, only A are held in for efficient initiation.

•the interactions are provided by various parts of polymerase holoenzyme, including σ region ¾ linker.• using an RNA polymerase w/ a mutant σ70 lacking σ region ¾

linker, requires much higher conc of initiating nucleotides.35

Mechanism of initial transcription• RNA pol translocated forward and synthesizes short transcripts

before aborting, repeats this cycle until escapes the promoter. • Three models:

• Transient excursions: RNA pol moves short along the DNA. return• Inchworming: only the front part moves (a flexible region)• Scrunching: pol remains stationary but pulls DNA in, ss bulges in it.

• Evidence support scrunching model.36

Promoter Escape•Promoter escape involves breaking polymerase-promoter and

polymerase cores-σ interactions.

•Abortive initiation: first, synth short RNA ~10 nt, transcripts are released, repeat this cycle again.

•Once RNA >~10 bp, stable complex formed. start elongation phase, until termination.

•the σ region ¾ as RNA mimic. This region lies in RNA exit channel in the open complex. If RNA >10 nt, this region must be ejected out, can take the enzyme several attempts.

•Scrunching is reversed upon escape. The DNA unwound during scrunching is rewound, concomitant collapse of the transcription bubble from 22-24 nt back down to 12-14 nt.

•Scrunching, a way to store and provides energy required tobreak polymerase-promoter and core-σ interactions associated with escape.

•The single-subunit RNA polymerase (as T7-RNA pol), lacking a σsubunit, undergoes comparable structurally shift in transition.

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(a) Untranslocated poly (0): RNA paired template for 9-nt stretch. (b) Forward translocated pol (+1), enzyme translocated 1-nt forward. (c) Forward translocated polymerase, with NTP bound: DNA/RNA in same

position, incoming NTP bound. (d) Reverse trans-located poly (-1), translocated backward 1-nt as during

hydrolytic editing. 38

Template and transcript in elongating complex

RNA core polymerase in the elongation phase

p.1027

The active site for transcription is in a cleft between

the and ’ subunits

’

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Sequences called terminators trigger the elongating

polymerase to dissociate from the DNA and release

the RNA chain. Terminators come in two types:

• Rho-independent terminator: polymerase to terminate

without the involvement of other factors.

• Rho-dependent terminator: requires an additional

protein Rho to induce termination.

Transcription Is Terminated by Signals

within the RNA Sequence

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Rho-independent terminator, or intrinsic terminators,

consists two elements: a short inverted repeat (about 20 nt

followed by a stretch of about eight A:T pairs). Function in the

RNA form, form the terminator hairpin in RNA.

Mutations that disrupt the terminator

Important for efficient termination

Hairpin cause termination by disrupting the elongation complex, by forcing open the RNA exit channel, or by disrupting RNA-template interactions

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Transcription termination

Rho-independent terminator :

(a) The hairpin forms in RNA as

soon as transcribed

(b) Hairpin structure disrupts

polymerase just as enzyme

transcribing the AT-rich stretch of

DNA down-stream.

(c) Exactly mechanism is not clear,

but weak interactions (Us in

transcript and As in template)

make release of that

transcript easier (A=U even

weaker than A=T).

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• less well-characterized RNA elements, requires Rho factor.

• Rho, ring-shaped 6 identical subunits, binds ssRNA as it exits the polymerase . The gap between subunits 1 and 6, is 12 Å , and helical pitch is 45 A.

• Rho, ATPase (RNA-DNA helicase): uses ATP to wrest the RNA from template and polymerase.

• How is Rho directed to an RNA molecule?

• some specificity in binding (rut sites, Rho utilization). Optimally, consist ~40 nt, largely ss-RNA, rich in CA residues.

Rho-Dependent Termination

• 2nd level of specificity, not to bind any transcript that being translated (coupled). In bacteria, Rho typically terminates only transcripts still being transcribed beyond the end.

Subunit Determines the Types of

Genes Expressed

different factor coordinate the expression of sets of genes to meet changes in cell physiology.

The availability of subunits depends on:regulated rates of synthesis and degradationPost-modifications ( between active inactive)anti- proteins to sequestering a specific sununit.

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The σ cycle

• The protein NusA (Mr 54,430) binds RNA polymerase competitively with σ subunit.

• Once transcription is complete, NusA dissociates from the enzyme and DNA, and a new σ factor can now bind and initiate transcription, in a cycle sometimes called the σ cycle.

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