Transcription: Chapter 13 Transcription Sections 13.1, 13.2, 13.4-13.13.8, 13.10-13.12.

Transcription: Chapter 13Transcription: Chapter 13

TranscriptionTranscription

Sections 13.1, 13.2, 13.4-Sections 13.1, 13.2, 13.4-13.13.8, 13.10-13.1213.13.8, 13.10-13.12


What is Transcription?What is Transcription?

The process of creating a transcript (RNA) The process of creating a transcript (RNA) from a template (DNA) from a template (DNA)

In Eukaryotes, this process occurs in the In Eukaryotes, this process occurs in the nucleus and is complex. This process is nucleus and is complex. This process is separate from Translation.separate from Translation.

In Prokaryotes, this process occurs in the In Prokaryotes, this process occurs in the cytoplasm of the cell and is less complex. cytoplasm of the cell and is less complex. Transcription is often coupled with Transcription is often coupled with Translation.Translation.


Key Players of TranscriptionKey Players of Transcription

RNA PolymeraseRNA Polymerase Promoter RegionPromoter Region Ribonucleoside Ribonucleoside

TriphosphateTriphosphate TemplateTemplate Rho (Rho ()) Transcription Transcription

FactorsFactors

CisCis -acting factors -acting factors TransTrans acting acting

factorsfactors


How Do We Know That RNA Is How Do We Know That RNA Is The Intermediate Between The Intermediate Between

DNA and Proteins?DNA and Proteins? DNA is located in the nucleus. DNA is located in the nucleus.

Proteins associated in the cytoplasm Proteins associated in the cytoplasm with ribosomes.with ribosomes.

RNA is chemically similar to DNA.RNA is chemically similar to DNA. RNA leave the nucleus and migrates RNA leave the nucleus and migrates

to cytoplasm.to cytoplasm. RNA is proportional to proteins RNA is proportional to proteins

present in the cytoplasmpresent in the cytoplasm


Transcription (Prokaryotes)Transcription (Prokaryotes)

RNA PolymeraseRNA Polymerase RNA polymerase (unwinds double helix)RNA polymerase (unwinds double helix) Ribonucleoside triphophates (U instead Ribonucleoside triphophates (U instead

of T)of T) No primerNo primer No proofreading abilitiesNo proofreading abilities Ribose (not deoxyribose)Ribose (not deoxyribose)


Points to PonderPoints to Ponder

Can you draw deoxyribose and ribose?Can you draw deoxyribose and ribose? Can you differentiate Uracil from Thymine?Can you differentiate Uracil from Thymine? Why is DNA considered a general and RNA Why is DNA considered a general and RNA

a soldier?a soldier? Can your draw a ribonucleoside Can your draw a ribonucleoside

triphosphate and a ribonucleoside triphosphate and a ribonucleoside monophosphate? Which one is the monophosphate? Which one is the precursor for transcription?precursor for transcription?


Transcription in Transcription in ProkaryotesProkaryotes



Prokaryotic gene is divided into three Prokaryotic gene is divided into three partsparts PromoterPromoter RNA-coding sequences is the DNA RNA-coding sequences is the DNA

sequence transcribed by RNA sequence transcribed by RNA polymerase into the RNA transcriptpolymerase into the RNA transcript

TerminatorTerminator



RNA Polymerase (RNA Polymerase (E. coliE. coli) – ) – , , , , ’’ subunits (500,000 Da) subunits (500,000 Da)

and and ’ active site for transcription’ active site for transcription Sigma (Sigma () subunit initiates RNA ) subunit initiates RNA

transcription by identifying promotertranscription by identifying promoter After 8 – 9 nucleotides assemble, After 8 – 9 nucleotides assemble, factor factor

dissociates from complexdissociates from complex Most common Most common subunit is subunit is 7070 ( (3232, , 5454, , ss, ,

EE do exist) do exist)


Promoter Region Promoter Region (Prokaryotes)(Prokaryotes)

TATAAT Box (-10 Region) – Pribnow TATAAT Box (-10 Region) – Pribnow box; box; ciscis acting factor acting factor

TTGACA (-35 Region) – GAC box; TTGACA (-35 Region) – GAC box; ciscis actingacting


Promoter consensus sequencesPromoter consensus sequences

Consensus TTGACA N16-17 TATAAT N6-7 A (+1)... gene

-35 box -10 box Start

TTTACA N17 TATGTT N6 A (+1)... geneLac operon

TTGACA N17 TTAACT N7 A (+1)... geneTrp operon

TTGATA N16 TATAAT N7 A (+1)... geneRecA

TTCCAA N17 TATACT N6 A (+1)... geneLexA

From: Fig. 12.4; Genetic Analysis and Principles 2nd Ed; Booker


Transcription: Initiation in Transcription: Initiation in E. E. colicoli

RNA Polymerase denatures DNA beginning RNA Polymerase denatures DNA beginning at the -10 boxat the -10 box

17 bp are denatured; forms transcription 17 bp are denatured; forms transcription bubblebubble

Open promoter complex is formed (ssDNA)Open promoter complex is formed (ssDNA) NTP’s begin to be inserted using +1 nt as NTP’s begin to be inserted using +1 nt as

the start site; sigma factor is released.the start site; sigma factor is released. Elongation beginsElongation begins


Transcription: Elongation in Transcription: Elongation in E. E. colicoli

RNA Polymerase denatures DNA as it RNA Polymerase denatures DNA as it moves moves

RNA Polymerase continues to covalently RNA Polymerase continues to covalently bond NTP’s to the growing RNA strandbond NTP’s to the growing RNA strand

RNA-DNA hybrid is formed for a short time RNA-DNA hybrid is formed for a short time after the RNA is produced; helps hold the after the RNA is produced; helps hold the complex together; structure in the RNA complex together; structure in the RNA Polymerase separates the DNA-RNA Polymerase separates the DNA-RNA hybrid; DNA re-annealshybrid; DNA re-anneals

Rate of elongation is 30-50 nt/secRate of elongation is 30-50 nt/sec



TerminationTermination Rho (Rho ()-independent termination – a )-independent termination – a

sequence with two-fold symmetry/self-sequence with two-fold symmetry/self-complementary about its center.complementary about its center.

Believed that the formation of the Believed that the formation of the hairpin loop within the transcript causes hairpin loop within the transcript causes destabilization of the RNA/DNA hybriddestabilization of the RNA/DNA hybrid


Transcription: Termination in Transcription: Termination in E. E. colicoli

Rho-independent termination or intrinsic Rho-independent termination or intrinsic terminationtermination Mediated by sequence elementsMediated by sequence elements Region that forms a stem loop followed by a uracil Region that forms a stem loop followed by a uracil

rich region near the end of the RNArich region near the end of the RNA After the RNA is synthesized, a stem loop formsAfter the RNA is synthesized, a stem loop forms Stem loop causes RNA pol to pauseStem loop causes RNA pol to pause U-rich region binds to the DNA; unstable; causes U-rich region binds to the DNA; unstable; causes

dissociation of DNA-RNA hybriddissociation of DNA-RNA hybrid Complex is release from the DNA; no protein Complex is release from the DNA; no protein

requiredrequired



Rho-dependent terminationRho-dependent termination Rho (protein) binds to the rut (rho utilization Rho (protein) binds to the rut (rho utilization

site) on the newly synthesized RNAsite) on the newly synthesized RNA Rho moves toward the RNA polRho moves toward the RNA pol A downstream portion of the newly synthesized A downstream portion of the newly synthesized

RNA forms a stem loopRNA forms a stem loop Stem loop causes RNA pol to pause (contacts Stem loop causes RNA pol to pause (contacts

RNA pol)RNA pol) Rho continues to move along the RNA and Rho continues to move along the RNA and

catches up with RNA polcatches up with RNA pol Rho acts as a helicase and breaks the H-bonds Rho acts as a helicase and breaks the H-bonds

between the DNA-RNA hybrid; dissociationbetween the DNA-RNA hybrid; dissociation


Transcription: Termination in Transcription: Termination in E. E. colicoli Rho-dependent terminationRho-dependent termination

Fig. 10.4; Genomes 2



Rho-dependant and Rho-independent Rho-dependant and Rho-independent terminationtermination Both involve a stem loop structure in the Both involve a stem loop structure in the

RNARNA Both involve the complete denaturation Both involve the complete denaturation

of the DNA-RNA hybrid and subsequent of the DNA-RNA hybrid and subsequent release of the RNA and RNA polymeraserelease of the RNA and RNA polymerase


Points to PonderPoints to Ponder

Why does the sigma factor Why does the sigma factor dissociates from the complex?dissociates from the complex?

What advantage does polycistronic What advantage does polycistronic RNA have in prokaryotes that RNA have in prokaryotes that monocistronic RNA does not have in monocistronic RNA does not have in eukaryotes?eukaryotes?

Why do you think ssBPs are not Why do you think ssBPs are not necessary in transciption but were necessary in transciption but were vital in replication?vital in replication?


Transcription in Transcription in EukaryotesEukaryotes


Transcription (Eukarytotes)Transcription (Eukarytotes)

Three different types of RNA polymerasesThree different types of RNA polymerases

RNA polymerase IRNA polymerase I – located in – located in nucleolus/rRNAnucleolus/rRNA

RNA polymerase IIRNA polymerase II – nucleoplasm/mRNA – nucleoplasm/mRNA RNA polymerase IIIRNA polymerase III – nucleoplasm/tRNA/ – nucleoplasm/tRNA/

5S rRNA 5S rRNA


Transcription: EukaryotesTranscription: Eukaryotes

Other differencesOther differences More complex (initiation and regulation)More complex (initiation and regulation)

More proteins are needMore proteins are need More regulatory elements in the DNAMore regulatory elements in the DNA

Extensive modification of the mRNAExtensive modification of the mRNA 3 different RNA polymerases3 different RNA polymerases


Why do you think Why do you think Transcription is more Transcription is more

complex in Eukaryotes complex in Eukaryotes than Prokaryotes?than Prokaryotes?

Transcription: Chapter 13Transcription: Chapter 13Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 11.7 Processes for synthesis of functional mRNA in Fig. 11.7 Processes for synthesis of functional mRNA in prokaryotes and eukaryotesprokaryotes and eukaryotes


Eukaryotic RNA Eukaryotic RNA polymerasespolymerases

RNA polymerases in Eukaryotes cannot RNA polymerases in Eukaryotes cannot bind directly to the DNA; all require bind directly to the DNA; all require protein transcription factors for initiationprotein transcription factors for initiation

Larger than bacterial counterparts and Larger than bacterial counterparts and have more subunitshave more subunits

All 3 Eukaryotic RNA Polymerase have All 3 Eukaryotic RNA Polymerase have similar structuresimilar structure 2 large subunits—have homology to beta and 2 large subunits—have homology to beta and

beta prime found in prokaryotes (what beta prime found in prokaryotes (what activity do they have?)activity do they have?)


Eukaryotic RNA Eukaryotic RNA polymerasespolymerasesRNA RNA

polymerasepolymerase SynthesizesSynthesizes LocationLocation

IIrRNA (28S, rRNA (28S, 18S, 5.8S)18S, 5.8S) nucleolusnucleolus

IIIIAll mRNAs All mRNAs and some and some snRNAssnRNAs

nucleoplasmnucleoplasm

IIIIIIAll tRNAs, 5S All tRNAs, 5S rRNA, other rRNA, other snRNAssnRNAs

nucleoplasmnucleoplasm


Transcription of mRNA in Transcription of mRNA in Eukaryotes: InitiationEukaryotes: Initiation

PromotersPromoters Basal promoter elements—components in the Basal promoter elements—components in the

DNA that are needed to get transcription DNA that are needed to get transcription started at low levels (promote basal started at low levels (promote basal transcription)transcription)

Proximal promoter elements—components Proximal promoter elements—components that are a distance from the promoter, but that are a distance from the promoter, but play a role in enhancing or suppressing play a role in enhancing or suppressing transcription; can influence the rate of transcription; can influence the rate of transcription or whether a gene is transcribed.transcription or whether a gene is transcribed.


Transcription of mRNA in Transcription of mRNA in eukaryotes: Initiationeukaryotes: Initiation

PromotersPromoters CisCis-acting (factors) elements—-acting (factors) elements—

components of the promoter that are components of the promoter that are located in the DNA. (TATA box, CAAT located in the DNA. (TATA box, CAAT box, GC box, enhancers, etc.)box, GC box, enhancers, etc.)

TransTrans-acting (factors) elements—-acting (factors) elements—components that bind to the DNA components that bind to the DNA (protein transcription factors, RNA (protein transcription factors, RNA polymerase, etc.)polymerase, etc.)



Promoters: Basal promoter elements Promoters: Basal promoter elements TATA box (element)TATA box (element)

Found at approximately -25Found at approximately -25 Consensus sequence is 5’-TATAAAA-3’Consensus sequence is 5’-TATAAAA-3’ Found in most eukaryotic promoters Found in most eukaryotic promoters Thought to be important for locating +1; when Thought to be important for locating +1; when

mutated the start site is not setmutated the start site is not set Important for basal transcriptionImportant for basal transcription Analogous to the -10 box in prokyotesAnalogous to the -10 box in prokyotes

Initiator element (Inr)—pyrimidine rich region Initiator element (Inr)—pyrimidine rich region near +1near +1


Can you think of 4 Can you think of 4 requirements for Basal-Level requirements for Basal-Level

Transcription?Transcription?


ANSWER:ANSWER:

RNA Polymerase, RNA Polymerase, Transcription Factors, Transcription Factors, Proteins and PromoterProteins and Promoter



Promoters: Proximal promoter Promoters: Proximal promoter elements elements Usually upstream of the promoter (approx Usually upstream of the promoter (approx

50-200 bp)50-200 bp) CAAT boxCAAT box

Found at approx -75Found at approx -75 GC boxGC box

Found at approx -90Found at approx -90 Can work in either orientationCan work in either orientation



Promoters: Regulatory elements—not required Promoters: Regulatory elements—not required for basal transcription for basal transcription Cis-acting elementsCis-acting elements Can be near the promoter or located great distances Can be near the promoter or located great distances

from the promoter/genefrom the promoter/gene May be upstream, downstream, or imbedded in the May be upstream, downstream, or imbedded in the

genegene May be enhancers (activator proteins bind here) to May be enhancers (activator proteins bind here) to

increase the rate of or activate transcription; a increase the rate of or activate transcription; a single one or severalsingle one or several

May be silencers/suppressors (suppressor proteins May be silencers/suppressors (suppressor proteins bind here); decrease the rate of or stop transcriptionbind here); decrease the rate of or stop transcription



RNA Polymerase cannot bind to the RNA Polymerase cannot bind to the DNA, so transcription factors must bind DNA, so transcription factors must bind firstfirst

TFIID binds to the TATA box—multi-TFIID binds to the TATA box—multi-subunitsubunit TBP protein—binds TATA boxTBP protein—binds TATA box TAF’s (at least 7)—binds TBPTAF’s (at least 7)—binds TBP Binds to basal promoter elements; role Binds to basal promoter elements; role

similar to sigma factor in bacteriasimilar to sigma factor in bacteria


Fig. 11.5 Events that may occur during the initiation of Fig. 11.5 Events that may occur during the initiation of transcription catalyzed by RNA transcription catalyzed by RNA

polymerase IIpolymerase II



TFIIB bind after TFIID is bound to the TFIIB bind after TFIID is bound to the promoterpromoter B associated with DB associated with D B recruits RNA pol II and TFIIF to the B recruits RNA pol II and TFIIF to the

promoterpromoter TFIID, B, and F form the minimal TFIID, B, and F form the minimal

promoter complexpromoter complex



TFIIE and H bind – makes initiation complex TFIIE and H bind – makes initiation complex completecomplete Produces the closed promoter complexProduces the closed promoter complex TFIIH important in promoting the open promoter TFIIH important in promoting the open promoter

complexcomplex Using ATP, H phosphorylates the CTD (carboxyl Using ATP, H phosphorylates the CTD (carboxyl

terminal domain of RNA pol IIterminal domain of RNA pol II Causes TFIIB to be released from the complexCauses TFIIB to be released from the complex H then uses its helicase activity to denature the DNAH then uses its helicase activity to denature the DNA TFIIE and H also dissociate when the open complex is TFIIE and H also dissociate when the open complex is

formedformed



The described process of initiation leads The described process of initiation leads to basal transcriptionto basal transcription

To regulate gene expression and/or the To regulate gene expression and/or the rate of transcription, a protein called the rate of transcription, a protein called the mediator links transcription factors that mediator links transcription factors that bind to regulatory factors and RNA pol IIbind to regulatory factors and RNA pol II

Mediator thought to affect CTD Mediator thought to affect CTD phosphorylation phosphorylation



Abortive transcription—a few Abortive transcription—a few nucleotides added in and nucleotides added in and transcription is stopped; not transcription is stopped; not understood why this occursunderstood why this occurs


Transcription of mRNA in Transcription of mRNA in Eukaryotes: Eukaryotes: Elongation/TerminationElongation/Termination Transition to elongation when RNA Transition to elongation when RNA

pol becomes processivepol becomes processive No termination signal/seq in euks; No termination signal/seq in euks;

termination is part of the termination is part of the modification of the mRNA (addition of modification of the mRNA (addition of poly(A) tail at the 3’ end of the mRNApoly(A) tail at the 3’ end of the mRNA


Genetic Code Genetic Code CharacteristicsCharacteristics

RNA derived from DNA; Nucelotides used RNA derived from DNA; Nucelotides used as “letters”as “letters”

3 Nucleotides = 1 Codon = 1 amino acid3 Nucleotides = 1 Codon = 1 amino acid 1 codon specifies 1 amino acid but 1 1 codon specifies 1 amino acid but 1

amino acid may have more than 1 codonamino acid may have more than 1 codon Start and Start codons presentStart and Start codons present No “breaks” between codonsNo “breaks” between codons


Genetic Code Genetic Code CharacteristicsCharacteristics

At one location within the mRNA is a At one location within the mRNA is a part of only one codonpart of only one codon

Code is universalCode is universal


Similarities between Similarities between Prokaryotic and Eukaryotic Prokaryotic and Eukaryotic

mRNA StructuremRNA Structure 5’ end—has a region that is not translated5’ end—has a region that is not translated

Called 5’ UTR (untranslated region)Called 5’ UTR (untranslated region) Variable lengthVariable length RegulatoryRegulatory

3’ end—has a region at the 3’ end that is 3’ end—has a region at the 3’ end that is not translatednot translated Called 3’ UTR (untranslated region)Called 3’ UTR (untranslated region) Variable lengthVariable length RegulatoryRegulatory



mRNA StructuremRNA Structure Coding region—part of the mRNA Coding region—part of the mRNA

that encodes the proteinthat encodes the protein Contains an AUG in the proper context Contains an AUG in the proper context

where translation startswhere translation starts Contains a stop codon in proper reading Contains a stop codon in proper reading

frame to end translationframe to end translation Length varies with the size of the Length varies with the size of the

polypeptidepolypeptide



mRNA StructuremRNA Structure

Transcription: Chapter 13 Transcription Sections 13.1, 13.2, 13.4-13.13.8, 13.10-13.12.

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Transcript of Transcription: Chapter 13 Transcription Sections 13.1, 13.2, 13.4-13.13.8, 13.10-13.12.