Gene lecture 11 transcription

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Flow of Genetic Information

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

The Genetic Code

The Discovery of the Genetic Code

 Linear form (mRNA derived from DNA)

 Francois Jacob and Jacques Monod (1961) – messenger RNA (mRNA)

 Triplet codons (triplets of ribonucleotides coding for 1 amino acid)

 Sydney Brenner (1960s) – codon in triplets (minimal use of the 4 mRNA bases to specificy 20 aa) (43=64)

 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)

 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

 Non-overlapping (single reading frame)  Universal (same ribonucleotide used by all organisms)

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)

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.

 RNA Mixed Copolymers

1A:5C (1/6 A: 5/6C)

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Repeating Copolymers

Results of Repeating Copolymers

 Developed by Gobind Khorana (1960s)  Synthetic long RNAs with repeating sequences

The Universal Genetic Code

Exceptions to the Universal Code

 Degeneracy  Wobble Hypothesis  Start codon (N-formylmethionine)

 Termination codons  Universal Viruses Bacteria Archaea Eukaryotes

Transcription  Uses DNA as a template  Catalyzed by RNA polymerase (holoenzyme of 500 kD) 

’ subunits

 Sense strand / template strand – DNA strand used as a template for transcription  Promoter region – DNA sequence recognized by  factor to initiate transcription (60 bases). (upstream of a gene)  TATA box (Pribnow box) – TATAAT sequence  Sigma factor (70, 28, 32, 54)

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 () 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.

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Eukaryotic Transcription  Features of eukaryotic transcription different from prokaryotic transcription:  Transcription inside the nucleus under the direction of 3 different RNA polymerases

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  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.

Trans -acting Factors  Transcription factors – facilitates template binding during the initiation of transcription  Example:  TFIID (TATA-binding protein or TBP) – binds to TATA-box

 Located 80 bases upstream from the start of transcription (-80)  Consensus sequence: GGCCAATCT  Influence the efficiency of the promoter

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

RNA Splicing  Ribozyme – RNA with catalytic activity  Self-excision process – process of RNA splicing or intron removal.  Transesterification – interaction between guanosine and the transcript.

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

 2 successive transesterification processes

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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)

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