Transcription Transcription

How the Information in DNA Is How the Information in DNA Is Used to Produce RNA in Used to Produce RNA in

Prokaryotes and EukaryotesProkaryotes and Eukaryotes

DNA

RNA

Protein

Information Flow From DNA

transcription

translation

replication

Components of RNA NucleotidesComponents of RNA NucleotidesPurine basesPurine bases Adenine (A)Adenine (A)

Guanine (G)Guanine (G)

Pyrimidine Pyrimidine basesbases

Cytosine (C)Cytosine (C)

Uracil (U)Uracil (U)

5-carbon sugar5-carbon sugar RiboseRibose

PhosphatePhosphate POPO44

RNA StructureRNA StructurePrimary Primary StructureStructure

Chain of Chain of

NucleotidesNucleotides

Secondary Secondary StructureStructure

Single folded chainSingle folded chain

RNA to RNA RNA to RNA base pairing rules:base pairing rules:G pairs with CG pairs with CA pairs with UA pairs with U

Base pairing creates the uniqueBase pairing creates the uniquesecondary structure of transfer RNA secondary structure of transfer RNA molecules.molecules.

TranscriptionTranscription

Base in DNABase in DNA

templatetemplate

Base in RNA Base in RNA productproduct

AA UU

TT AA

GG CC

CC GG

Transcription = production of RNA using DNA as Transcription = production of RNA using DNA as a template a template

One DNA strand is used to produce a RNA strand, One DNA strand is used to produce a RNA strand, beginning at a promoter and ending at a terminator. beginning at a promoter and ending at a terminator.

Structure of Transcribed RegionStructure of Transcribed Region

Transcription Transcription

Transcription requires Transcription requires 1. RNA polymerase, an enzyme that adds 1. RNA polymerase, an enzyme that adds nucleotides in a 5’nucleotides in a 5’3’ direction. 3’ direction. 2. Nucleoside triphosphates2. Nucleoside triphosphates 3. Energy: release of diphosphate3. Energy: release of diphosphate

3’---TACAAAGAGACT---5’3’---TACAAAGAGACT---5’

5’---ATGTTTCTCTGA ---3’ 5’---ATGTTTCTCTGA ---3’

3’---TACAAA GAGACT---5’ DNA template3’---TACAAA GAGACT---5’ DNA template

5’---ATGTTTCTCTGA---3’5’---ATGTTTCTCTGA---3’

5’---AUGUUUCUCUGA---3’ mRNA

Antisense strandAntisense strand

Sense strandSense strand

Different Genes Can Have Different Genes Can Have Different Template StrandsDifferent Template Strands

For each gene, only one of the two For each gene, only one of the two DNA strands is used as a template. DNA strands is used as a template.

Applying Your KnowledgeApplying Your Knowledge

What is the sequence of an RNA molecule What is the sequence of an RNA molecule transcribed from a DNA template transcribed from a DNA template strand that reads 3’-ATG-5’?strand that reads 3’-ATG-5’?

1.1. 5’-TAC-3’5’-TAC-3’2.2. 5’-CAU-3’5’-CAU-3’3.3. 5’-AUG-3’5’-AUG-3’4.4. 5’-UAC-3’5’-UAC-3’5.5. 3’-TAC-5’3’-TAC-5’

Stages of TranscriptionStages of Transcription

• InitiationInitiation– transcription apparatus binds to promotertranscription apparatus binds to promoter

• ElongationElongation– RNA polymerase adds nucleotides to chainRNA polymerase adds nucleotides to chain

• TerminationTermination– recognition of termination signalrecognition of termination signal– separation of RNA from DNA templateseparation of RNA from DNA template

Features of the Prokaryotic PromoterFeatures of the Prokaryotic Promoter

Two Forms of RNA Polymerase Two Forms of RNA Polymerase in Prokaryotesin Prokaryotes

Core enzyme Core enzyme

continuescontinuestranscription transcription after initiationafter initiation

Holoenzyme = Core enzyme Holoenzyme = Core enzyme + Sigma factor + Sigma factor Recognizes –10 and –35 regionsRecognizes –10 and –35 regionsBinds to promoterBinds to promoterForms open promoter complex Forms open promoter complex

InitiationInitiation

Holoenzyme forms

Holoenzyme binds to promoter

Open promoter complex forms

First RNA nucleotide binds

ElongationElongation

RNA nucleotides are added in a 5’RNA nucleotides are added in a 5’3’ direction 3’ direction by RNA polymerase core enzyme.by RNA polymerase core enzyme.

Rho-Dependent TerminationRho-Dependent Termination

•Rho-Dependent terminators Rho-Dependent terminators have have

•region without secondary region without secondary structure structure•sequence that produces a sequence that produces a pause in transcription pause in transcription

•Rho protein binds to regionRho protein binds to region without secondary structure without secondary structure

•Terminator sequences form Terminator sequences form hairpin to slow polymerase hairpin to slow polymerase

•Rho travels to terminator regionRho travels to terminator region and unwinds RNA-DNA duplex and unwinds RNA-DNA duplex

Rho-Independent Rho-Independent TerminationTermination

•Rho-independent terminators haveRho-independent terminators have•inverted repeatsinverted repeats•string of adeninesstring of adenines

•When terminator is transcribedWhen terminator is transcribed•inverted repeats form hairpininverted repeats form hairpin that slows RNA polymerase that slows RNA polymerase•A-U pairs are unstableA-U pairs are unstable

•RNA separates from templateRNA separates from template

Eukaryotic TranscriptionEukaryotic Transcription

Eukaryotic RNA Eukaryotic RNA PolymerasePolymerase

Messenger RNA Messenger RNA (mRNA)(mRNA)

RNA Polymerase IIRNA Polymerase II

Ribosomal RNA Ribosomal RNA (rRNA)(rRNA)

RNA Polymerase IRNA Polymerase I

Transfer RNATransfer RNA

(tRNA)(tRNA)

RNA Polymerase IIIRNA Polymerase III

Different types of RNA are produced Different types of RNA are produced by different polymerases.by different polymerases.

Features of the Eukaryotic RNA Features of the Eukaryotic RNA

Polymerase II PromoterPolymerase II Promoter

General Transcription Factors +General Transcription Factors +RNA Polymerase II bind to Core PromoterRNA Polymerase II bind to Core Promoter

Transcriptional Activator ProteinsTranscriptional Activator Proteins bind to the Regulatory Promoter bind to the Regulatory Promoter

Initiation of Eukaryotic Polymerase II TranscriptionInitiation of Eukaryotic Polymerase II Transcription

Transcription Factor IID binds to Transcription Factor IID binds to TATA box on core promoterTATA box on core promoter

Holoenzyme forms and binds to TFIID Holoenzyme forms and binds to TFIID Holoenzyme = RNA Pol II + Transcription Factors Holoenzyme = RNA Pol II + Transcription Factors

Basal Transcription

Apparatus

Initiation of Eukaryotic Initiation of Eukaryotic Polymerase II TranscriptionPolymerase II Transcription

•Basal Transcription Apparatus is needed toBasal Transcription Apparatus is needed to support minimal levels of transcription. support minimal levels of transcription. •Transcription can be increased by Transcription can be increased by Transcriptional Activator Proteins bound to Transcriptional Activator Proteins bound to

•Regulatory PromoterRegulatory Promoter•Enhancer SequenceEnhancer Sequence

Termination of Eukaryotic Termination of Eukaryotic Polymerase II TranscriptionPolymerase II Transcription

• Polymerase II continues Polymerase II continues transcription beyond the transcription beyond the coding regioncoding region

• Cleavage occurs at the Cleavage occurs at the 3’ end, near consensus 3’ end, near consensus sequence sequence

• Rat1 Rat1 exoexonuclease binds nuclease binds to 5’ end and moves to 5’ end and moves toward Polymerase II, toward Polymerase II, degrading the RNA from degrading the RNA from 5’5’3’3’

• Transcription terminates Transcription terminates when Rat1 reaches when Rat1 reaches Polymerase IIPolymerase II

exonuclease

Transcription Termination for Eukaryotic Transcription Termination for Eukaryotic RNA Polymerases I and IIIRNA Polymerases I and III

• RNA Polymerase I requires a RNA Polymerase I requires a termination factor that binds to a DNA termination factor that binds to a DNA sequence downstream of the sequence downstream of the termination sitetermination site

• RNA Polymerase III transcribes a RNA Polymerase III transcribes a terminator sequence to produce a region terminator sequence to produce a region rich in uracil residues on the transcriptrich in uracil residues on the transcript

Applying Your KnowledgeApplying Your Knowledge

Which organism(s) haveWhich organism(s) haveA. Sequences at –10 and –35 that are A. Sequences at –10 and –35 that are

recognized by Holoenzyme?recognized by Holoenzyme?B. Transcriptional Activator Proteins? B. Transcriptional Activator Proteins? C. Rho-Dependent Termination?C. Rho-Dependent Termination?D. Rat1 exonuclease activity?D. Rat1 exonuclease activity?

1.1. ProkaryotesProkaryotes2.2. EukaryotesEukaryotes3.3. Both Prokaryotes and EukaryotesBoth Prokaryotes and Eukaryotes4.4. Neither Prokaryotes nor EukaryotesNeither Prokaryotes nor Eukaryotes