Medical Genetics-Transcription and Translation Robert F. Waters, PhD DNA hRNA mRNA rRNA tRNA

DNA Helical Structure Base Pairing Chromatin/Chromosomes Operon

Protein Synthesis-Overview Major classes of RNA mRNA Prokaryotic and Eukaryotic Eukaryotic (hnRNA – mRNA) Leader (Header) region 5’ untranslated region (5’ UTR) Eukaryotic cap region attached 7-methylguanylate Trailer region 3’ untranslated region (3’ UTR) Amount of mRNA About 5% in prokaryotes Stability mRNA lasts for just a few minutes

RNA Ribosomal RNA (rRNA) Prokaryotic Three kinds 23s rRNA (2904 nucleotides)—component of 50s rRNA subunit 16s rRNA (1541 nucleotides)—part of small 30s rRNA subunit 5s rRNA (120 nucleotides)—part of 50s rRNA subunit Abundance – about 80% of all RNA

RNA Continued: Ribosomal RNA Eukaryotic Abundance 28s (60s subunit) Transcription product of separate gene Abundance 4% -- 45s 71% -- full rRNA

RNA Continued: tRNA (Transfer RNA) Prokaryotic Average about 80 nucleotides Some common structure so may function with rRNA tRNAs derived from larger precursor tRNAs Abundance (about 15% of total RNA in prokaryotes)

RNA Continued: tRNA (Transfer RNA) Eukaryotic (Very similar to prokaryotic) Average about 80 nucleotides Some common structure so may function with rRNA tRNAs derived from larger precursor tRNAs Abundance (about 15% of total RNA in prokaryotes)

Other RNAs Small RNAs Eukaryotes Small Cytoplasmic RNAs (scRNAs) Signal recognition particles Small Nuclear RNAs (snRNAs) Snurps Spliceosome Separation of introns and exons

Transcription Formation of RNA from DNA Nucleus Single strand of DNA acts as template Substrates ATP GTP CTP UTP Note cancer chemotherapy

Direction of RNA Synthesis RNA chain grown proceeds from 5’ to 3’ Codon sequence Therefore amino acid sequence Not understood as to which DNA strand

Elongation Enzymes in Transcription Prokaryotes DNA Dependent RNA Polymerase Core enzyme is trimeric Necessary for RNA elongation Holoenzyme has core enzyme with another subunit required for initiation Eukaryotes Many large enzymes usually greater than 500000 MW Called Type I, II, III

Promoters of Transcription Prokaryotic Promoter sequences Initiation site (startpoint) Pribnow Box (similar to TATA box) TATAAT 9-18 BP upstream from initiator -35 sequence 15 BP upstream from Pribnow Box TTGACA

Promoters of Transcription Eukaryotic promoters May be a purine or pyrimidine Usually purine in prokaryotes Multiple promoter sequence Order of boxes TATA Box (25 BP from Initiator) CCAAT (CAAT BOX) GGGCG (GC BOX) NOTE: RNA Polymerase III promoters occur downstream of the startpoint! Between +8-+30 and +50-+70

Initiation of Transcription Prokaryotes  (sigma) factor Allows RNA polymerase holoenzyme to attach to promotor sequences Process  (sigma) factor facilitates opening of DNA Enzyme (holoenzyme) forms phosphodiester bond between first two bases Elongation begins and after 10 nucelotides have been added,  factor is released Released  factor can bind with another holoenzyme and initiate transcription at some other location

Initiation of Transcription Eukaryotic Initiation Factors Need four (4) factors to initiate transcription from a TATA Box region TFIID Binds directly to the TATA Box promoter region TFIIA Binds with TFIID that is already bound to TATA Box TFIIB Facilitates RNA Polymerase II binding to aggregate TFIIE Binds to preinitiation complex and triggers beginning of transcription at initiation point (start point)

Termination of Transcription Prokaryotes Factor-independent termination Inverted repeat causing a loop Loop area rich in GC pairs Stability for slow down or stop Stretch of Uracils (poly U) after loop region Factor-dependent termination Certain sequences act as termination sequences in presence of Rho-factor (). Eukaryotes Not much known Transcription does continue several BP beyond pseudo-termination with Poly A (Tail region)

Post-transcriptional Processing in Eukaryotes Formation of 5’ Cap Cap 0 – methyl group Cap 1 – two BP Cap 2 – three BP Poly adenylation (Tail) Sliceosome SNURPS Intronic excision Contiguous exons

Protein Synthesis (Translation) Sequence of triplet code form AA sequence on RER Genetic Code mRNA (CODON) Redundancy 64 possible codes 20 Aas Stop Codons (UAA, UAG, UGA)

Activation of tRNAs Coupling of AAs to tRNA Aminoacyl-tRNA synthetases Activate Amino Acids and facilitate attachment to 3’ end of tRNA Associated with anti-codon Wobble-base Repair of improper associated tRNA

Eukaryotic Ribosomes 80s from a 60s and 40s subunits 60s subunit A-site (Aminoacyl Site) P-site (peptidyl transfer site) T-site (Termination site)