Transcription

RNA Structure Composition of RNA in metabolically active cell Ribose: both 2’ and 3’ OH Uracil(U) instead of thymine (T) Mostly single strand Secondary structure Intrastrand base-pairing Composition of RNA in metabolically active cell mRNA: 3~5 % rRNA: 90% tRNA: 4%

RNA Polymerase RNA synthesis using ssDNA as a template Prokaryote One RNA polymerase Eukaryote RNA Pol I rRNA (28S, 5.8S, 18S) RNA Pol II mRNA RNA Pol III tRNA, 5S rRNA 5’ to 3’ RNA synthesis

Transcription and Translation in Prokaryote * Promoter region --- RNA polymerase binding site * Termination sequence

Genes Gene Specific nucleotide sequence that is transcribed into RNA Structural gene Gene encoding protein Polycystronic transcription Prokaryote only Transcription of multiple genes in one transcription unit -35 -10 Operator ORF1 ORF2 ORF3 Terminator promoter mRNA

Transcription and Translation in Eukaryote Exon: 150~ 200 bp Intron: 40 ~ 10,000 bp (intervening sequence)

Splicing of a Eukaryotic Primary RNA Transcript

Alternative Splicing Generation of tissue-specific proteins from the same structural gene Different pattern of exon splicing from a primary transcript

A small number of eukaryotic structural genes lack intron (e. g A small number of eukaryotic structural genes lack intron (e.g. olfactory genes). Exon-skipping mechanism generates different gene products in different tissues from the same structural gene.

Translation

Initiation of Translation in Prokaryotes (1) mRNA + small ribosomal subunit ↑Ribosome binding site (Shine-Dalgarno [SD] sequence) (2) + fMet-tRNAfMet (f: formyl) (3) + large ribosomal unit

tRNA anticodon (tRNA, 5’-CAU-3’)  codon (mRNA, 5’-AUG-3’)

Genetic Code Information in DNA  amino acid sequence in protein mRNA 5’ GCA GCA CUA GGA GAG AAG 3’ Codon: triplet of RNA bases 20 amino acids in nature Codon (nt) Amino acid 1 4 2 16 3 64

Genetic Code

Initiation of Translation in Eukaryotes (1) Met-tRNAMet + small ribosomal subunit (along with initiation factors) (2) + 5’ end of mRNA  The complex migrates along the mRNA until an AUG is encountered. (3) + large ribosomal subunit

Translation Initiation Elongation Termination Elongation and termination phases are very similar in prokaryotes and eukaryotes.

Elongation Phase of Translation 5’3’ direction at a rate of about 15 amino acids per sec. A single mRNA can be translated simultaneously by a number of ribosomes

Table 3.2 Genetic code and codon usage

Termination stop (termination) codon ~ UAA, UAG, UGA no tRNAs which bind to stop codons Protein(s) (termination factor, or release factor) binds to the ribosome.

In most proteins, the methionine at the N-terminus is cleaved off, leaving the second encoded amino acid as the N-terminal moiety.

Amino Acids and Primary Structure Amino group Carboxyl group R group; 20 Side chains Peptide bond Between NH2 and COOH Polypeptide A chain of amino acids N terminus and C terminus

Amino Acids

Regulation of Transcription in Bacteria

Operon Promoter region Frequently, bacterial structural genes that encode proteins for a single metabolic pathway are contiguous. This arrangement is called an operon. Promoter region -10 region (TATAAT, TATA box, Pribnow box) -35 region (TTGAC sequence)

Negative controlled system Repressor Induction of the on state Effector molecule (derepressor) Induction of the off state IR: inactive repressor, aporepressor C: corepressor

Positive controlled system Activator Promoting RNA polymerase activity Repressed by an effector molecule

Regulation of Transcription in Eukaryotes

Unlike the situation in prokaryotes, operons are almost never found in the genomes of eukaryotes. In addition to DNA-protein interactions, protein-protein associations are important for regulating eukaryotic transcription. Promoter region -25 bp (TATA box, or Hogness box) 8 nucleotides including TATA sequence -75 bp (CCAAT sequence, “cat” box) -90 bp (GC box --- repeated GC)

Promoter region

Transcription initiation complex