Transcription

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Transcription

• AHMP 5406

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Objectives

• Describe the general process of DNA
  transcription
• Discuss the function of RNA polymerases
• Differentiate between messenger RNA, small
  nuclear RNA, ribosomal RNA, and transfer RNA
• Explain the purpose of the promoter and
  terminator
• Compare and contrast the three types of RNA
  polymerase in eukaryotic nuclei RNA polymerase
  I, RNA polymerase II, and RNA polymerase III
• Discuss RNA capping RNA splicing
• Explain the selective exportation of mRNAs from
  the nucleus
• Discuss ribosomal RNA and its function

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What is Transcription ?

• DNA does not direct protein synthesis directly
• RNA is used as intermediate
• Transcription Copying DNA into RNA

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RNA

• Ribonucleic Acids
• Contain ribose instead of deoxyribose (DNA)
• Polymers formed by A, G, C, and U
• U uracil
• Complementary to Thymine
• But sometimes binds to G
• Single stranded
• Can bind to itself and form 3D structures

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DNA to RNA

• Transcribed RNA is complementary to 1 DNA strand
• Transcribe RNA transcript
• Transcripts can only reach a few thousand bases
  in length
• Unidirectional
• 5-3

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RNA Types

• Coding
• Messenger RNA (mRNA)
• Non-coding
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• Small nuclear RNA (snRNA)
• Small nucleolar RNA (snoRNA)

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mRNA

• Codes for proteins
• Can be modified by splicing, 5 and 3 additions

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Non-Coding RNAs

• rRNA
• Form basic structure of ribosome
• tRNA
• Adaptors b/w mRNA and AA
• snRNA
• Several nuclear processes
• Assist in splicing
• snoRNA
• Process and chemically modify rRNA

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DNA sequences used as signals

• Sequences signal transcription to start and stop
• Promoters start signals
• s factor binds to promoter in bacteria
• Terminators stop signals
• In bacteria A-T region causes fold

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RNA polymerases

• RNA pols perform transcription
• Immediate release of transcript allows many RNA
  to be synth. in a short time
• More error prone than DNA pols
• They have limited error correction abilities

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Types of RNA polymerases

• RNA pol I
• Transcribe 5.8S, 18S and 28S genes
• S Svedberg units, sedimentation coefficient
  related to centrifugation
• The bigger the number the bigger the molecule
• RNA pol II
• Transcribe protein-coding genes, snoRNA genes
• Some snRNA genes
• RNA pol III
• Transcribe tRNA genes and some snRNA genes

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Transcription in Euks.

• Requires general transcription factors to
  initiate transcription
• GTFs help position RNA pol II correctly
• TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
• Recognize specific sites on DNA

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RNA pol start points

• TATA Box promoter
• Recognized by subunit of TFIID
• TBP TATA Box Binding protein
• approx 25 bp upstream of initiation point
• Causes conformational change in DNA

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Other necessary proteins for initiation of
transcription

• Transcriptional activators
• Attract RNA pol II to start site
• Mediators
• Allow communication between activator and GTFs
• Chromatin remodeling proteins
• Modify DNA conformation

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Elongation Factors

• Increase affinity of RNA Pol II to DNA
• Assist in moving through chromatin structure

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mRNA modifications

• 5 capping
• Splicing removing introns
• 3 Polyadenylation

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5 Capping

• When RNA Pol II at 25 bases 5 cap is added to
  transcript
• Modified guanine
• Three enzymes
• Phosphatase removes phosphate from 5 end
• Guanyl transferase adds GMP
• Reversed linkage 5-5
• Methyltransferase adds methyl group to guanosine

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Splicing

• Removes introns (or sometimes shuffling)
• Sequences signal where to cut
• Spliceosome performs cutting
• Complex of snRNAs (Us) and protein

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Self Splicing Mechanisms

• Group I
• Bind free G nucleotide to specific site
• Group II
• Uses reactive A nucleotide
• Unusual mechanisms

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Poly A tail

• Cleavage stimulating factor F (CstF)
• Cleavage and polyadenylation specificity factor
  (CPSF)

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Export of mRNA

• Goes through nuclear pore complexes
• Mature mRNA are modified and protein bound
• Signal (passport) for transport of mRNA outside
  of nucleus
• 5 cap proceeds first

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rRNAs

• Transcribed by RNA Pol I
• rRNAs are made from a larger precursor
• Sections are modified, cleaved and assembled into
  ribosomes in nucleolus

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Subnuclear structures

• Sites where snRNA processing machinery is
  assembled, stored and recycled
• Cajal bodies
• GEMS (Gemini of coiled-bodies)
• Interchromatin granule structures