Gene Lecture 11 Transcription
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Fundamental Genetics Lecture 9
The Genetic Code and Transcription John Donnie A. Ramos, Ph.D. Dept. of Biological Sciences College of Science University of Santo Tomas
Flow of Genetic Information
1
The Genetic Code Linear form (mRNA derived from DNA) Triplet codons (triplets of ribonucleotides coding for 1 amino acid) Unambiguous (1 codon = 1 amino acid only) Degenerate ( 1 amino acid can be specified by several codons) Contains specific start and stop codons Commaless (no breaks once translation starts until the stop codon is reached) Non-overlapping (single reading frame) Universal (same ribonucleotide used by all organisms)
The Discovery of the Genetic Code Francois Jacob and Jacques Monod (1961) – messenger RNA (mRNA) Sydney Brenner (1960s) – codon in triplets (minimal use of the 4 mRNA bases to specificy 20 aa) (43=64) Francis Crick – frameshift mutations alters the codons Mariane Manago and Severo Ochoa polynucleotide phosphorylase (synthesis of RNA without template)paved the way to the production of RNA polymeres in cell free-systems
2
The Discovery of the Genetic Code Marshall Nirenberg and J. Heinrich Matthaei (1661) – codons used cell-free protein synthesizing system and polynucleotide phosphorylase RNA Homopolymers (UUUUUU…, AAAAAAA…, CCCCCC…, GGGGG…) UUU (Phenylalanine) AAA (Lysine) CCC (Proline)
RNA Mixed Copolymers
1A:5C (1/6 A: 5/6C)
The Triplet Binding Assay Developed by M. Nirenberg and P. Leder (1964) Mimics the in vivo translation of proteins where a mRNA-tRNAribosome complex is formed when all three macromolecules are allowed to interact.
3
Repeating Copolymers Developed by Gobind Khorana (1960s) Synthetic long RNAs with repeating sequences
Results of Repeating Copolymers
4
The Universal Genetic Code Degeneracy Wobble Hypothesis Start codon (N-formylmethionine)
Termination codons Universal Viruses Bacteria Archaea Eukaryotes
Exceptions to the Universal Code
5
Transcription Uses DNA as a template Catalyzed by RNA polymerase (holoenzyme of 500 kD)
αββ’σ subunits
Sense strand / template strand – DNA strand used as a template for transcription Promoter region – DNA sequence recognized by σ factor to initiate transcription (60 bases). (upstream of a gene) TATA box (Pribnow box) – TATAAT sequence Sigma factor (σ70, σ28, σ32, σ54)
Transcription RNA polymerase don’t need primers Elongation in 5’ to 3’ direction Rate in E coli: 50 bases/sec at 37°C Termination is a function of rho (ρ) factor – hexameric protein interacting with the end of a gene Polycistronic mRNA – bacterial mRNA containing information for the synthesis of proteins of related function Monocystronic mRNA – eukaryotic mRNA containing information for a single protein.
6
Eukaryotic Transcription Features of eukaryotic transcription different from prokaryotic transcription: Transcription inside the nucleus under the direction of 3 different RNA polymerases
Presence of protein factors (promoters, enhancers, etc.) binding to the upstream portion of a gene (cis-acting elements) during initiation step. Presence of post-transcriptional regulation.
Cis -acting Elements TATA Box (Goldberg-Hogness Box) Located 30 bases upstream from the start of transcription (-30) Consensus sequence: TATAAAA Facilitates denaturation of helix because it is ATrich region
CAAT Box Located 80 bases upstream from the start of transcription (-80) Consensus sequence: GGCCAATCT Influence the efficiency of the promoter
7
Trans -acting Factors Transcription factors – facilitates template binding during the initiation of transcription Example: TFIID (TATA-binding protein or TBP) – binds to TATA-box
Post-transcriptional Processing 7-methylguanosine cap (7mG) Protection from nucleases Role mRNA transport across the nuclear membrane
3’ cleavage site: AAUAAA Failure of 3’ cleavage results to absence of poly A tail
Split genes – contains intervening sequences
8
RNA Splicing Ribozyme – RNA with catalytic activity Self-excision process – process of RNA splicing or intron removal. Transesterification – interaction between guanosine and the transcript. 2 successive transesterification processes
The Spliceosome Alternative splicing Small nuclear ribonucleoproteins (snRNP or snurps) – bonds to GU or AG sites of introns 2 transesterification processes Snurps form a loop (lariat) in the branch point region Produces isoforms of proteins
9
RNA Editing Substitution editing changes in the nucleotide bases of a given mRNA Common in mitochondrial RNA and chloroplast RNA Example: Apoliprotein B (Apo B) – C to U change CAA to UAA
Insertion / deletion editing addition or removal of nucleotide sequences Common in mitochondrial RNA or guide RNA (gRNA)
10
The Genetic Code and Transcription John Donnie A. Ramos, Ph.D. Dept. of Biological Sciences College of Science University of Santo Tomas
Flow of Genetic Information
1
The Genetic Code Linear form (mRNA derived from DNA) Triplet codons (triplets of ribonucleotides coding for 1 amino acid) Unambiguous (1 codon = 1 amino acid only) Degenerate ( 1 amino acid can be specified by several codons) Contains specific start and stop codons Commaless (no breaks once translation starts until the stop codon is reached) Non-overlapping (single reading frame) Universal (same ribonucleotide used by all organisms)
The Discovery of the Genetic Code Francois Jacob and Jacques Monod (1961) – messenger RNA (mRNA) Sydney Brenner (1960s) – codon in triplets (minimal use of the 4 mRNA bases to specificy 20 aa) (43=64) Francis Crick – frameshift mutations alters the codons Mariane Manago and Severo Ochoa polynucleotide phosphorylase (synthesis of RNA without template)paved the way to the production of RNA polymeres in cell free-systems
2
The Discovery of the Genetic Code Marshall Nirenberg and J. Heinrich Matthaei (1661) – codons used cell-free protein synthesizing system and polynucleotide phosphorylase RNA Homopolymers (UUUUUU…, AAAAAAA…, CCCCCC…, GGGGG…) UUU (Phenylalanine) AAA (Lysine) CCC (Proline)
RNA Mixed Copolymers
1A:5C (1/6 A: 5/6C)
The Triplet Binding Assay Developed by M. Nirenberg and P. Leder (1964) Mimics the in vivo translation of proteins where a mRNA-tRNAribosome complex is formed when all three macromolecules are allowed to interact.
3
Repeating Copolymers Developed by Gobind Khorana (1960s) Synthetic long RNAs with repeating sequences
Results of Repeating Copolymers
4
The Universal Genetic Code Degeneracy Wobble Hypothesis Start codon (N-formylmethionine)
Termination codons Universal Viruses Bacteria Archaea Eukaryotes
Exceptions to the Universal Code
5
Transcription Uses DNA as a template Catalyzed by RNA polymerase (holoenzyme of 500 kD)
αββ’σ subunits
Sense strand / template strand – DNA strand used as a template for transcription Promoter region – DNA sequence recognized by σ factor to initiate transcription (60 bases). (upstream of a gene) TATA box (Pribnow box) – TATAAT sequence Sigma factor (σ70, σ28, σ32, σ54)
Transcription RNA polymerase don’t need primers Elongation in 5’ to 3’ direction Rate in E coli: 50 bases/sec at 37°C Termination is a function of rho (ρ) factor – hexameric protein interacting with the end of a gene Polycistronic mRNA – bacterial mRNA containing information for the synthesis of proteins of related function Monocystronic mRNA – eukaryotic mRNA containing information for a single protein.
6
Eukaryotic Transcription Features of eukaryotic transcription different from prokaryotic transcription: Transcription inside the nucleus under the direction of 3 different RNA polymerases
Presence of protein factors (promoters, enhancers, etc.) binding to the upstream portion of a gene (cis-acting elements) during initiation step. Presence of post-transcriptional regulation.
Cis -acting Elements TATA Box (Goldberg-Hogness Box) Located 30 bases upstream from the start of transcription (-30) Consensus sequence: TATAAAA Facilitates denaturation of helix because it is ATrich region
CAAT Box Located 80 bases upstream from the start of transcription (-80) Consensus sequence: GGCCAATCT Influence the efficiency of the promoter
7
Trans -acting Factors Transcription factors – facilitates template binding during the initiation of transcription Example: TFIID (TATA-binding protein or TBP) – binds to TATA-box
Post-transcriptional Processing 7-methylguanosine cap (7mG) Protection from nucleases Role mRNA transport across the nuclear membrane
3’ cleavage site: AAUAAA Failure of 3’ cleavage results to absence of poly A tail
Split genes – contains intervening sequences
8
RNA Splicing Ribozyme – RNA with catalytic activity Self-excision process – process of RNA splicing or intron removal. Transesterification – interaction between guanosine and the transcript. 2 successive transesterification processes
The Spliceosome Alternative splicing Small nuclear ribonucleoproteins (snRNP or snurps) – bonds to GU or AG sites of introns 2 transesterification processes Snurps form a loop (lariat) in the branch point region Produces isoforms of proteins
9
RNA Editing Substitution editing changes in the nucleotide bases of a given mRNA Common in mitochondrial RNA and chloroplast RNA Example: Apoliprotein B (Apo B) – C to U change CAA to UAA
Insertion / deletion editing addition or removal of nucleotide sequences Common in mitochondrial RNA or guide RNA (gRNA)
10
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