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• 28s (60s subunit)• 18s (40s subunit)• 5.8s (60s subunit)• 5s (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)