day 3 - transcription and rna processing

7/30/2019 Day 3 - Transcription and RNA Processing

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Transcription and RNA Processing

The Central Dogma

For the phenotypic expression of any gene, information contained inDNA is first transcribed into an RNA from where it is translated intothe amino-acid sequence of the corresponding protein. Phenotypesare the manifestation of the activity/function of proteins and

catalytic RNAs.

DNA RNATranscription

RNA Pol + factors

ProteinTranslation

Ribosomes, tRNAaa+ other factors

Function

Enzyme, structuralelement, hormone,

regulatory, etc

The assumptions of the central dogma aresound, but there are exceptions!

Phenotype


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The Revised Central Dogma

DNA

REVERSE

TRANSCRIPTION Ribozymes

FUNCTION

FUNCTION

Interactions


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Exceptions to the Central Dogma

1. Information contained in RNA can be copied into DNA by reversetranscriptases and telomerase. EXAMPLES: retroviruses, mobileintrons.

2. After transcription, nucleotides can be added to, deleted from, or

modified in some mRNAs by processes known as RNA editing.EXAMPLES: addition and deletion of Us in the mitochondria oftrypanosomes, conversion of Cs to Us in plant mitochondria bydeamination.

3. Not all genes are expressed phenotypically through proteins.EXAMPLES: the RNAs of mobile introns are nucleases, the LSUribosomal RNA has peptide synthetase activity. Such RNAs areclassified as ribozymes.

Ribozymes favor the concept that a more primitiveRNA-based ancestral form of life may have preceded

the DNA-, RNA-, protein-based life as we know it now.


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The first step of the expression of a gene (DNA) is to transcribe theinformation contained in the nucleotide sequence from one strand(template or antisense strand) into an RNA. The product of thistransaction is a transcript.

If a gene carries information for the assembly of a protein, thetranscript is a messenger RNA (mRNA).

The information in mRNAs is decoded by ribosomes and translated byalignment of three-nucleotide codons with corresponding tRNAsthat deliver the corresponding amino acids.

Transcription

Translation


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The messenger: a generalview of transcription and

translation

U UU UU

Coding strand of DNA

Template strand of DNA

mRNA 5 3


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Transcription andRNA ProcessingTopics

1. RNA structure.

2. RNA synthesis.3. Transcription: initiation, elongation and

termination.

4. RNA processing: cleavage of precursortranscripts and modification of bases.

5. RNA and protein splicing.

6. RNA editing.Videos
http://www.hhmi.org/biointeractive/dna/animations.htmlhttp://www.hhmi.org/biointeractive/dna/animations.html


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Ribonucleoside triphosphate

O

Base

RNA precursors

Fig. 2.1

RNA polynucleotide chain

5 3


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RNA Structure

Secondary structure: regions of

base pairing (double-strandedhelical regions)

Stem-loop Fig 2.2

Primary structure: order of the nucleotides read 5

35ACUCAUCGGCACGUCAUGCUGAUAUCCGGCUUGACACU 3

Tertiary structure: folding of theentire RNA chain

Pseudoknot

Only non-covalent hydrogenbonding is involved in

secondary and tertiarystructure formation


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Tertiary structure of a transfer RNA

5

3


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Structure ofE. coli16S rRNA

1. ~1500 nucleotides long

2. Compact 3-D folding

3. Highly conserved

5

3


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Kinds of RNA found in a typical bacterium (E. coli)

RNA Function SizeStability

T

No. of

differentkinds

mRNAMessenger contains information forassembly of amino-acid sequence of

protein by ribosomes and tRNA

6-

20S

Few

minutes

~4000

tRNATransfer codon recognition inpartnership with ribosome and transferof amino acid into polypeptide

4S

longerthan one

generation

variable


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RNAs and DNAs can be separated by size in sucrosegradients by centrifugation and by gel-electrophoresis

16S23S30S

30S Precursor

23S16S

5S

+

Start

Separation byElectrophoresis


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Transcription: RNA polymerase (RNAP) catalyzedsynthesis of RNA on a DNA template

Do you remember the principles of nucleic-acid synthesis? Ch. 1!

RNA polymerasesdo NOT require aprimer for theinitiation of RNA

synthesis.

Direction of RNAsynthesis:

5 3

Direction of readingof template DNAstrand:

3

5


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SizeSubunit aa Da Gene Function

alpha () 329 36,511 rpoA Assembly of RNAP, binding of someregulatory proteins, catalysis.

beta () 1342 150,616 rpoB Catalysis of chain initiation and

elongation.

beta () 1407 155,159 rpoC Binding to DNA template.

omega () 91 10,237 rpoZ Restores denatured enzyme to fully

functional form.sigma (70) 613 70,263 rpoD Directs enzyme to corresponding

promoters.

Subunits of the E. coli holo RNAP and

their Functions


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Transcription starts at promoters, located in the DNAupstream of the coding region of one gene or agroup of functionally related genes.

Important Features of Transcription inProkaryotes

Promoters are relatively short nucleotide sequences inthe DNA (genome) that are recognized by sigma ()

proteins (factors). Sigma factors bound topromoters recruit (interact, bind) the RNApolymerase core enzyme for the initiation of

transcription at specific transcription start sites thatare part of the promoter sequence in the DNA.

What do

promoterslook like?


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Typical structure of Bacterial Housekeeping-Gene70

Promoters with 10 and 35 Regions

Question: What do the negative numbers mean?

Fig 2.6

Number of bases upstream (5) of a designated place, which, in thiscase, is the first base of the RNA (transcription start point).

DNA CodingTemplate

RNA (A/G)NNNNN --

53

Coding-strandconsensus sequences


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RNA exitchannel

Core RNA polymerase Holoenzyme

Active site of RNA polymerase and binding of70

Promoter contacts

10 35

pincer

pincerActive sitechannel

Secondary channelfor nucleosidetriphosphate entry

Crabclaw structure

1 4 are domains of70 proteins


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Proteins called factors bind to coreRNAP and direct it to DNA nucleotidesequences called promoters. The twostrands of the DNA are separated by . Atranscription bubble is generated, andtranscription starts at a T or C in thetemplate strand. No primer is requiredfor transcript initiation and no helicase isrequired for the unwinding of the DNA.

RNA synthesis proceeds for ~8 9 ntalong the template, is released.

Transcription starts at promotersand ends at terminators

Fig. 2.7

Transcription continues until thepolymerase with its transcription bubblereaches a transcription termination (t)site. The RNA and the polymerase

dissociate and are released from theDNA.


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Closed complex(RPC)

Open complex(RPO)

binding

Promoter recognition

DNA isomerization

Escape

release and elongation

Termination

AbortInitiation

Transcription elongationcomplex (TEC)

Transcription Cycle


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Not all 70 promoters are created equal

UP element

Extended 10


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Evolutionarily Conserved Regions andFunctional domains of Bacterial 70 Factors

Inhibition of-DNA binding

70 factors initiate the transcription of housekeeping genes (namedafter the 70 kDa housekeeping factor ofE. coli).

Shown below is the linear arrangement of the 70-like factor ofThermus

aquaticus(~43 kDa).

Down arrows indicate contact points with core RNAP proteinsElectrostatic interactionsDirect hydrogen bondsIndirect hydrogen bonds

potential hydrophobic (van der Waals) contacts.

From S. Borukhov and E. Nudler 2003. Curr. Opinion. Microbiol. 6: 93-100


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RNAP Holoenzyme Open Initiation Complex

Template-strand

Coding-strand

Nascent RNA

Part of has been removed to expose the interior of the holoenzymeinitiation complex.

Modified from K.S. Murakami andS. A. Darst 2003. Curr. OpinionStructural Biol. 13: 31-39.

RNA channel


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Transcription Elongation Complex (TEC)

The RNA polymerase adds from 30 to 100 nucleotides per second to thegrowing RNA molecule (6000 maximum per min).


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RNA Polymerase BacktrackingThe formation of a secondary structure (stem-loop or hairpin) at the 3

end of a growing transcript can cause backtracking of the RNApolymerase. The 3 end of the RNA is pushed into the secondary channel.GreA/GreB insert their N-termini, which have exonuclease activity, intothe secondary channel and degrade the RNA until it is rearranged(properly paired with the DNA template?) in the active site channel so that

elongation can be resumed. The true function of RNAP pausing andbacktrackingis somewhatenigmatic.


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The number of different factors

varies greatly among organismsMycoplasma genitaliumhas only ONE factor

E. coli has SEVEN different factorsStreptomyces coelicolorhas MORE THAN SIXTYdifferent factors

Each type offactor recognizes its own uniqueclass of promoter sequences

See nextpage


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The Seven Sigma Factors ofE. coliandtheir Functions

Sigmafactor Gene Function

70 (D) rpoD Principal sigma factor (housekeeping genetranscription).

N (54) rpoN(ntrA, glnF) Nitrogen-regulated gene transcription.

H

(32

) rpoH Heat-shock gene transcription.S (38) rpoS Gene expression in stationary phase cells.

E (24) rpoE Periplasmic stress response proteins.

F (28) rpoF Expression of flagellar operons.FecI fecI Regulates the fecgenes for iron dicitrate

transport.


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70 (D) TTGACA 16 18 TATAAT

H (32) CTTGAA 13 15 CCCCATNT

E (24) GAACTT 15 17 TCTGA

F (28) CTAAA 15 GCCCATAA

FecI (18) GAAAAT 15 TGTCCT

S (38) TTGACA 14 18 CTAYACTT

CONSENSUS SEQUENCESigma 35 region Spacer (b) 10 region

25 region Spacer (b) 12 region

N (54) CTGGCAC 6 TTGCA

Consensus Sequences ofE. coliPromoters


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Transcription is not a smoothly flowing process: pausing occursfrequently at sites that allow the formation ofhairpin structures in theRNA. When hairpins structures form in the portion of the RNA that islocated in the exit channel of the polymerase they cause temporarydisplacement of the 3 OH from the polymerization active site of the RNApolymerase. When this happens, the polymerase backtracks along thetemplate, and several nucleotides at the 3 OH end are pushed into thesecondary channel, where they are progressively removed backward byGreA and GreB until a proper pairing of the RNA with the DNA template is

restored. Pausing is an important feature involved in the regulation ofgene expression through coordination of the synthesis of mRNA with itssimultaneous translation, termination and antitermination oftranscription, and attenuation of transcription.

Watch for signals along the track!


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GC-rich inverted repeat

There are two kinds of transcription termination:1. Factor independent termination and2. Factor-dependent termination.

Transcription Termination

Factor-independent transcription termination occurs at sites in the DNAthat include a region of two-fold symmetry followed by a stretch ofatleast four As in the template strand.

Fig. 2.18MORE

Run ofat least 4As inthe template strand


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RNA

RNA 3 end

Factor-independent transcription termination

Dissociation of nascent RNAand RNA polymerase from

template strand

Fig. 2.18

Folding of the transcript (RNA) in

the active site channel breakshydrogen bonding to thetemplate DNA strand and causesthe release of the RNA and the

core RNA polymerase.

MORE


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Factor-independent transcription termination

How did scientist figure out this mechanism of termination?

Mutations that disrupt base pairing in the hairpin loop structure of RNA(two-fold symmetry in DNA) or shorten the run of adjacent Us (As inthe DNA template) cause continuation of transcription beyondterminator sites.


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Factor-dependent Transcription Termination

Features of factor-dependent transcription-termination sites in DNA: a

site specifying a sequence in the RNA, for example rut, that isrecognized and bound by a protein, for example Rho () that chasesthe RNA polymerase and releases it and the transcript from the DNAtemplate at transcription pausing sites (usually a G:C-rich sequence).

IMPORTANT: Factor-dependent transcription occurs preferentially whenthe translation of a nascent mRNA is stalled.

DNA

Polysomes

DNA

In prokaryotes, translation iscoupled to transcription.

Specifies factor-binding sequence inRNA (EXAMLE:rut)

Any transcription-pause

site in DNA


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Factor-dependent transcription termination

Fig 2.19

How does it work?Rho hexamer binds to rutin thenascent RNA and then chasesthe RNA polymerase until itreaches it at a transcription-pause site on the DNA. Rhothen unwinds the RNA/DNAduplex, releasing the transcriptand the RNA polymerase from

the DNA.

RNA polymerasereaches a pausesite

Stalledribosome

bindsrut

unwindsRNA/DNA hybrid

RNA and RNAP arereleased


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Rho () binds at rutsequences in nascent RNA, moves along RNA(movement requires ATP) until it reaches the RNA polymerase at atranscription-pause site. HOWEVER, if a ribosome has passed a rutsiteBEFORE is bound, then the ribosome prevents from catching up withthe RNA polymerase, and transcription continues past the pause-site.Rho appears to be an RNA/DNA helicase. However, it is not clearwhether or not can directly access the RNA/DNA duplex within theactive-site channel of a paused RNA polymerase core enzyme.

The other two proteins that have termination-factor characteristics similarto those of are:

Tau () and NusA

In comparison to , the RNA-binding sites and interactions ofTau andNusA with the RNA polymerase at transcription pause sites are not wellcharacterized yet.

E. coli has at least three differenttranscription-termination factors


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Antibiotics that Inhibit Transcription

RifampinRifampin is a member of the rifamycins,macrocyclic lactone antibiotics that

inhibit transcription at the initiationstage, but do not block elongation onceinitiation is complete. Rifamycins bindto the -subunit in the wall of theactive-site channel of the RNAP ofbacteria, mitochondria andchloroplasts. Two or three nucleotidesare polymerized.

MORE


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Action of Rifampin

pppApN and pppGpN are the mostcommon products.

The Streptovaricins are related compounds that have the same action as

rifampin, except that they also can block transcript elongation.


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Actinomycin DBinds to the major groove of DNA in G/C rich regions.

Inhibits transcription and replication

Antibiotics that Inhibit Transcription


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Think about it!

How does the process of RNA synthesis differ from DNA synthesis withrespect to:

1. Substrates?

2. Initiation?

3. Template?

4. Priming?5. Ancillary enzymes?

6. Termination?

7. Editing?


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RNA Processing

RNA Modification

and

RNA Editing

i d ( )


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RNA Processing: rRNA and tRNA (rRNA operon)

Transcript (unprocessed precursor RNA)

Fig. 2.20

16 S tRNA 23S 5S

Further processing and modification of bases (maturation)

MATURE rRNAs and tRNAs

Endonucleolytic cleavages by Rnases III, P, etc.

Processed RNA products

SpacerSpacer


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tRNAprocessing

Rnase P

RNase

RNase RNase

rRNAprocessing

in E. coliandB. subtilis

Rnase M5 is similarto type II DNA

topoisomerases

Rnase P consists of an RNA dimer(same sequence, catalytic subunit)

complexed with a dimer of a smallprotein. It is a ribozyme.

difi i


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Structure of a mature tRNA

RNA modification

U

Fig. 2.21

The most common RNAmodification is U

Added by CCAtransferase

IV

II

I

III

Determining base

Dihydrouridine

D d ti f RNA


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Degradation of mRNA

Some of the

enzymesinvolved alsoparticipate inrRNA and tRNAprocessing

Box 2.5

1 1500 l tid


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16S rRNA

1. ~1500 nucleotids

2. Many modified bases

3. Compact 3-D folding4. Complexed with 21

proteins

5. Highly conserved

RNA i


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Group I introns

Group II introns

Introns in eukaryotic mRNAs

RNA processingIntrons and splicing

Splicing: Removal of parasiticDNA information from RNA

Box 2.6


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Removal of

parasitic DNAinformation fromproteins

Removal of inteins

GyrA of many prokaryotes

contains an intein

Box 2.6

VMA1 protein of yeast

Intein

1 284 738 1071

N-extein C- extein

454

RNA Editing: Edited ND1 mRNA of T brucei * deleted U


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uGAUACAAAAAAACAUGACUACAUGAUAAGUAuCAuuuuAuGuuAuuuuuGGuAGuuuuuuuACAuu

uGuAuCGuuuuACAuuuG*GUCCACAGCAuCCCG***CAGCACAuG**GuGuuuuAuGuuGuuuAuuGuA

uuuuuGuGGuGA*AuuuAuuGuuuA**UAUUGAuUGuAuuAuA***G*GuuAUUUGCAUCGUGGUACAG

AAAAGUUAUGUGAAUAUAAAAGUGUAGAACAAUGUCUUCCGuAUUUCGACAGGUUAGAuuAuG

uuA*GuGuuuGuuGuAAuGAGCAuuuGuuGuCuuuA***UGuuuuGAGuAuAuGuuGCGAuGuuGuuuGu

CGuuACGuuGuGCAuuuAuGCGuuuAuuAAuuGuA****GAAuuuAC***CCGuAGuuuuAAuGGuuuGuuGuGuAuAuCAuGuAuGGuuuuGG*AuuuAGGuuGuuuGuCUCCGuuG*UUAuGAuCAuuuGAGGAA***

CG*UGACAAAuuGAuGACAuuuuuuGAuuuAuG**UUGuGGuuGuCGuAuGCAuuuGGCUUUCAuGGu

uuuAuuA*GGuAUUCUUGAUGAuuuuGuuuuuGGuuuuGuuGAuuuuuuGuuGuuGuuGA***UAAuAuC

AuGuuuGuuuGuuAuGGAuuGuuAuGAuuuGuuAuuuGuGGGuAAUCGuuuAuuuUAuuuGCGuuuGC***GuGGuuuGuCAuuuuuuGAuuuAuAuGAuuuA**GuuuuuA**A**UAGuuuAAGuGGuGuuuuGuCuCGu

uCGuuAGGuAuGGuGuGAGAuuGUCGuuuAuuuAGuuGuuA****UGA*****GuUGuAuuuuAuGuuuuG

uuAuGAuuAuuGuuuuuGuuuuAuAGGuGAuGCAuuuGA*UCGuuuAuuuuuACGuuuGuuuGAUAuGC

GuAuGAGuuuGuuGAuuuGuAAGCAAuGuuuuuuuGuuGGuuuuuuuGuuuuuG*****GuuuuGuuuGuuuGuuuG**AuuAuuuAuAuuGuGAuAuuACCAuuG****AGACCAuuAuuAuGuuAuuuuAuAGuuuGuGGu

GuuGuuGuuuGCCGGGuAuA*UCAuuuGC*UUGUGuuGAACACCCCAAAGGuGA***GuAuuGuuuGu

uAuuA****UGuuuuuGuGuuGGuuuAuGuuCUCGuuuACGuuuGCGuuGuGCGGAuuuuuuGCA*UAUU

UGuuuAuuGGAuGuuuGuuuGCGuGGuuuuuuAuuGCAuGAuuuAGuuGC***C*GuuuuAGGuAAuAuuGAuGuuGuuuuuGGAuCCGUAGAUCGuuA*GuuuuAuAuGuG**A******GGUUAUUGuAGGAUUGUU

UAAAAUUGAAUAAAAA

RNA Editing: Edited ND1 mRNA ofT. bruceiu added U

Courtesy of Dr. Donna Koslowsky

Mitochondrial RNA Editing in Trypanosomes


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Mitochondrial RNA Editing in Trypanosomes

Editing a substrate RNA by an editosome using a guide RNA

Substrate mRNAs are transcribed from mtDNA maxicircles (5-6)Guide RNAs are transcribed from mtDNA minicircles (~1000)

Modified from Catteneo (1990)

AUAUAAAAGCGGGAGUUAUUUUUAUUAUUUUUU 3

UAAAAGUAAUAAA5

A

G CC

C

A

C

C

C

A

AAAA

G

U

UUUUUUUUU

U

UU UU

* .... .. .... ..

5

3

AnchorTether

GUIDE

5

ATATAAAAGCGGGAGTTA A A 3DNA

Transcript

Guide RNA

Edited segment

EDITOSOME


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Mitochondrial and Chloroplast RNAs

Untranslated regions (UTRs) and secondary structuremodifications of nuclear RNAs in eukaryotes

RNA Editing: Base Modifications

C U Editing in the co 2 mRNA of mai e mitochond ia


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C U Editing in the cox2 mRNA of maize mitochondria

day 3 - transcription and rna processing

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