TranscriptionTranscription

AHMP 5406AHMP 5406

Objectives:Objectives:1.1. Describe the general process of DNA transcriptionDescribe the general process of DNA transcription

2.2. Discuss the function of RNA polymerasesDiscuss the function of RNA polymerases

3.3. Differentiate between messenger RNA, small nuclear RNA, ribosomal Differentiate between messenger RNA, small nuclear RNA, ribosomal RNA, and transfer RNARNA, and transfer RNA

4.4. Explain the purpose of the promoter and terminatorExplain the purpose of the promoter and terminator

5.5. Compare and contrast the three types of RNA polymerase in Compare and contrast the three types of RNA polymerase in eukaryotic nuclei: RNA polymerase I, RNA polymerase II, and RNA eukaryotic nuclei: RNA polymerase I, RNA polymerase II, and RNA polymerase IIIpolymerase III

6.6. Discuss RNA capping & RNA splicingDiscuss RNA capping & RNA splicing

7.7. Explain the selective exportation of mRNAs from the nucleusExplain the selective exportation of mRNAs from the nucleus

8.8. Discuss ribosomal RNA and its functionDiscuss ribosomal RNA and its function

What is What is Transcription Transcription ??

DNA does not direct protein DNA does not direct protein synthesis directlysynthesis directly

RNA is used as RNA is used as intermediateintermediate

Transcription = Copying Transcription = Copying DNA into RNADNA into RNA

RNARNA Ribonucleic AcidsRibonucleic Acids

Contain ribose instead of Contain ribose instead of deoxyribose (DNA)deoxyribose (DNA)

Polymers formed by A, G, C, and Polymers formed by A, G, C, and UU

U = uracilU = uracil Complementary to ThymineComplementary to Thymine But sometimes binds to GBut sometimes binds to G

Single strandedSingle stranded Can bind to itself and form 3D Can bind to itself and form 3D

structuresstructures

DNA to RNADNA to RNA

Transcribed RNA is Transcribed RNA is complementary to 1 DNA complementary to 1 DNA strandstrand

Transcribe RNA = Transcribe RNA = transcripttranscript

Transcripts can only reach Transcripts can only reach a few thousand bases in a few thousand bases in lengthlength

UnidirectionalUnidirectional 5’-3’5’-3’

RNA TypesRNA Types

CodingCoding Messenger RNA (mRNA)Messenger RNA (mRNA)

Non-codingNon-coding Ribosomal RNA (rRNA)Ribosomal RNA (rRNA)

Transfer RNA (tRNA)Transfer RNA (tRNA)

Small nuclear RNA (snRNA)Small nuclear RNA (snRNA)

Small nucleolar RNA (snoRNA)Small nucleolar RNA (snoRNA)

mRNAmRNA

Codes for proteinsCodes for proteins

Can be modified by splicing, 5’ and 3’ Can be modified by splicing, 5’ and 3’ additionsadditions

Non-Coding RNAsNon-Coding RNAs

rRNA rRNA Form basic structure of ribosomeForm basic structure of ribosome

tRNAtRNA Adaptors b/w mRNA and AAAdaptors b/w mRNA and AA

snRNAsnRNA Several nuclear processesSeveral nuclear processes Assist in splicingAssist in splicing

snoRNAsnoRNA Process and chemically modify rRNAProcess and chemically modify rRNA

DNA sequences used as signalsDNA sequences used as signals

Sequences signal Sequences signal transcription to start and transcription to start and stop stop

Promoters – start signalsPromoters – start signals factor binds to promoter factor binds to promoter

in bacteriain bacteria

Terminators – stop Terminators – stop signalssignals In bacteria A-T region In bacteria A-T region

causes foldcauses fold

RNA polymerasesRNA polymerases

RNA pols perform transcriptionRNA pols perform transcription

Immediate release of transcript allows Immediate release of transcript allows many RNA to be synth. in a short timemany RNA to be synth. in a short time

More error prone than DNA polsMore error prone than DNA pols

They have limited error correction abilitiesThey have limited error correction abilities

Types of RNA polymerasesTypes of RNA polymerases

RNA pol IRNA pol I Transcribe 5.8S, 18S and 28S genesTranscribe 5.8S, 18S and 28S genes S = Svedberg units, sedimentation coefficient related S = Svedberg units, sedimentation coefficient related

to centrifugationto centrifugation The bigger the number the bigger the moleculeThe bigger the number the bigger the molecule

RNA pol IIRNA pol II Transcribe protein-coding genes, snoRNA genesTranscribe protein-coding genes, snoRNA genes Some snRNA genesSome snRNA genes

RNA pol IIIRNA pol III Transcribe tRNA genes and some snRNA genesTranscribe tRNA genes and some snRNA genes

Transcription in Euks.Transcription in Euks.

Requires general transcription Requires general transcription factors to initiate transcriptionfactors to initiate transcription

GTFs help position RNA pol II GTFs help position RNA pol II correctlycorrectly

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIHTFIIH

Recognize specific sites on DNARecognize specific sites on DNA

RNA pol start pointsRNA pol start points

TATA Box promoterTATA Box promoter Recognized by subunit of TFIIDRecognized by subunit of TFIID TBP – TATA Box Binding proteinTBP – TATA Box Binding protein approx 25 bp upstream of initiation pointapprox 25 bp upstream of initiation point Causes conformational change in DNACauses conformational change in DNA

Other necessary proteins for Other necessary proteins for initiation of transcriptioninitiation of transcription

Transcriptional activatorsTranscriptional activators Attract RNA pol II to start Attract RNA pol II to start

sitesite

MediatorsMediators Allow communication Allow communication

between activator and between activator and GTFsGTFs

Chromatin remodeling Chromatin remodeling proteinsproteins

Modify DNA conformationModify DNA conformation

Elongation FactorsElongation Factors

Increase affinity of RNA Pol II to DNAIncrease affinity of RNA Pol II to DNA

Assist in moving through chromatin Assist in moving through chromatin structurestructure

mRNA modificationsmRNA modifications

5’ capping5’ capping

Splicing = removing Splicing = removing intronsintrons

3’ Polyadenylation3’ Polyadenylation

5’ Capping5’ Capping

When RNA Pol II at 25 bases When RNA Pol II at 25 bases 5’ cap is added to transcript5’ cap is added to transcript

Modified guanineModified guanine

Three enzymesThree enzymes Phosphatase = removes Phosphatase = removes

phosphate from 5’ endphosphate from 5’ end Guanyl transferase = adds GMPGuanyl transferase = adds GMP

Reversed linkage 5’-5’Reversed linkage 5’-5’ Methyltransferase = adds methyl Methyltransferase = adds methyl

group to guanosinegroup to guanosine

SplicingSplicing

Removes introns (or Removes introns (or sometimes shuffling)sometimes shuffling)

Sequences signal where to Sequences signal where to cutcut

Spliceosome performs Spliceosome performs cuttingcutting Complex of snRNAs (Us) and Complex of snRNAs (Us) and

proteinprotein

Self Splicing MechanismsSelf Splicing Mechanisms

Group IGroup I Bind free G nucleotide to Bind free G nucleotide to

specific sitespecific site

Group IIGroup II Uses reactive A nucleotideUses reactive A nucleotide

Unusual mechanismsUnusual mechanisms

Poly A tailPoly A tail

Cleavage stimulating factor F Cleavage stimulating factor F (CstF)(CstF)

Cleavage and polyadenylation Cleavage and polyadenylation specificity factor (CPSF)specificity factor (CPSF)

Export of mRNAExport of mRNAGoes through nuclear pore complexesGoes through nuclear pore complexes

Mature mRNA are modified and protein boundMature mRNA are modified and protein bound

Signal (passport) for transport of mRNA outside Signal (passport) for transport of mRNA outside of nucleusof nucleus

5’ cap proceeds first5’ cap proceeds first

rRNAsrRNAs

Transcribed by RNA Pol ITranscribed by RNA Pol I

rRNAs are made from a rRNAs are made from a larger precursorlarger precursor Sections are modified, Sections are modified,

cleaved and assembled into cleaved and assembled into ribosomes in nucleolusribosomes in nucleolus

Subnuclear structuresSubnuclear structures

Sites where snRNA Sites where snRNA processing machinery is processing machinery is assembled, stored and assembled, stored and recycledrecycled Cajal bodies Cajal bodies GEMS (Gemini of coiled-GEMS (Gemini of coiled-

bodies)bodies) Interchromatin granule Interchromatin granule

structuresstructures