MBG 2040 Lecture Notes - Lecture 8: Small Nuclear Rna, Central Dogma Of Molecular Biology, Francis Crick
2 May 2018
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Transcription and RNA Processing
Outline:
• Transfer of genetic information: the central dogma
• Process of gene expression
• Transcription in prokaryotes
• Transcription and RNA processing in eukaryotes
Central Dogma of Molecular Biology
• First articulated by Francis Crick in 1958
• Understanding of these fundamental biological processes has earned many Nobel prizes
• Central Dogma:
1. Perpetuation of genetic information from generation to generation
• DNA undergoes replication via DNA dependent DNA polymerase
2. Control of the phenotype: gene expression
• Transcription via DNA-dependent RNA polymerase
▪ *reverse transcription via RNA-dependent DNA polymerase (reverse
transcriptase)
• Translation (mRNA --> polypeptide) is a complex process involving ribosomes,
tRNAs, and other molecules
Process of Gene Expression
• In prokaryotes, transcription and translation occur at the same time (they are coupled)
o *see figure on slide
• Transcription, translation and mRNA degradation often occur simultaneously in prokaryotes
• Gene expression in eukaryotes:
o Transcription and translation are not coupled in eukaryotes
o Transcripts must be transported to the cytoplasm for translation
• *see slide
• Types of RNA:
o Messenger RNA (mRNA) - intermediates that carry genetic information from DNA to the
ribosomes
o *Transfer RNA (tRNA) - adaptors between amino acids and the codons in mRNA
o *Ribosomal RNA (rRNA) - structural and catalytic components of ribosomes
o *Small nuclear RNA (snRNAs and snoRNAs) - spliceosomes and rRNA, tRNA modifcation,
respectively
o *Micro RNAs (miRNAs, siRNA, RNAi) - short single-stranded RNAs that block expression
of complementary mRNAs
*note: many RNAs do not encode protein
• *see figure: gene expression and the different roles of RNA in eukaryotes
• Transcription in a chemical reaction:
o Need:
• DNA template
• 4 ribonucleotide triphosphates (rNTPs) - A,U,C,G
• DNA dependent RNA polymerase
o (rNMP)n + rNTP --> (rNMP)n+1 + Ppi
o *see figure on slide
• 5' to 3' direction of chain growth (DNA template is read from 3' to 5')
• DNA template
▪ 3' end is non-template strand
▪ 5' end is template strand
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• General Features of RNA synthesis:
o Similar to DNA synthesis except that…
• Precursors are ribonucleotide triphosphates (rNTPs)
• Only one strand of DNA is used as a template
• RNA chains can be initiated de novo (no primer required)
o RNA molecule will be complementary to the DNA template (anti-sense) strand and identical
(except that uridine replaced thymidine) to the DNA non-template (sense) strand
o RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5' to 3' direction
Transcription in Prokaryotes:
• Steps:
o RNA chain initiation
• RNA polymerase binds, unwinds DNA and joins first two nucleotides
o RNA chain elongation
• Complementary nucleotides continue to be added
o RNA chain termination
• Transcription stops --> nascent RNA molecule
• E. coli RNA polymerase
o Tetrameric core: alpha2, beta, beta' - transcribes any DNA
o Holoenzyme: alpha2, beta, beta', sigma - transcribes specific genes
o Functions of subunits:
• Alpha - assembly of tetrameric core
• Beta - ribonucleoside triphosphate binding site
• Beta' - DNA template binding region
• Sigma - initiation of transcription specifically at promoter
• *see model of transcription based on x-ray diffraction data
• Process (in typical E. coli):
o Initiation of transcription:
• Sigma factor binding site at -35 element
▪ 5' TTGACA 3'
• Localized unwinding at -10 element (very A/T rich)
▪ 5' TATAAT 3' (Pribnow box)
• Transcription initiate about 5-9 base pairs doen from the end of the -10 sequence
• The 5' end of RNA is usually a purine (+1 position)
o Transcription elongation - the transcription bubble
• ~17 base pairs are unwinded
• RNA polymerase has helix unwinding and rewinding activities
o Termination (rho-independent)
• Transcript is released when RNA polymerase pauses after hair pin formation (at
AAAAA --> UUUUU *area of weak hydrogen binding)
▪ This weak H-bonding at U:A residues allows mRNA release from DNA when
RNA polymerase pauses at terminator
• *note: 3' end of RNA transcript (UUUUU) has -OH
Transcription and RNA Processing in Eukaryotes:
• Puffs (Balbiani rings) in Drosophila polytene salivary chromosomes are sites of localized
unwinding due to gene transcription
• Most eukaryotes and three RNA polymerases:
o RNA polymerase I
• Located in nucleolus
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