PROTEIN SYNTHESIS: TRANSCRIPTION + TRANSLATION Anna Thomassen Program • DNA vs. RNA • Transcription: Prokaryotic genes • Transcription: Eukaryotic genes • Post-transcriptional modifications • Translation • Translation Eukaryotic vs. Prokaryotic genes • Post-translational modifications • Questions DNA vs. RNA: The Importance of Oxygen... DNA vs. RNA: The Importance of meth(yl group)... CH3 DNA vs. RNA The Importance of T and U... ;) DNA vs. RNA DNA (Deoxyribose RNA (Ribose Nucleic Acid) Nucleic Acid) Basic Structure of Sugar, phosphate, N-base Nucleotides Length Much longer than RNA Much shorter than DNA Sugar Deoxyribose Ribose - Extra Oxygen Nitrogen base Thymine Uracil • Additional methyl group Strands Double-stranded Single-stranded (some exceptions – looks like A-DNA) Stability More stable Less stable - Stores genetic material in - Transfers genetic information permanent way RNA = Ribonucleic Acid Ribosomal RNA (rRNA) Transfer RNA (tRNA) Messenger RNA (mRNA) = Components of = Transfers amino acids = Carries genetic information ribosomes to mRNA for assembly of amino acids • Sizes: 23S, 16S, 5S into proteins • Smallest: 4S • Sizes: 28S, 18S, 5.8S, 5S • 80% of total 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ PROTEIN SYNTHESIS Transcription – Post-transcriptional modifications – Translation – Protein processing Making protein TRANSCRIPTION = synthesis of RNA Location • Prokaryotes - cytoplasm • Eukaryotes - nucleus STEPS OF TRANSCRIPTION: Initiation, Elongation, Termination Transcription video Prokaryotic RECIPE • Enzymes TRANSCRIPTION: Prokaryotic genes • RNA Polymerase • DNA Topoisomerase Initiation Inhibited by • Promoter • RNA Polymerase recognizes + binds to promotor Actinomycin D • -35 sequence -35 sequence + Pribnow box (-10) (! Not transcribed) • 5’-TTGACA-3’ • Majority of gene expression is controlled Inhibited by Rifampicin • Pribnow box • 5’-TATAAT-3’ Elongation RNA produced: 5’ – 3’ • Formation of transcription bubble – local unwinding of short stretch of DNA DNA template strand: 3’ – 5’ • Generation of supercoils DNA topoisomerases Transcription goes on while • Sigma subunit is released translation occurs (no • DNA-RNA hybrid helix is formed nucleus) • RNA polymerase slides along template DNA strand (3’→ 5’ direction on DNA), complementary bases pair up RNA polymerase links nucleotides to 3’-end of mRNA • Continues until termination signal is reached Termination: 2 processes • Intrisic/Rho-independent: hair-loop formation • Rho-Dependent Prokaryotic Transcription Termination • Rho-independent (Intrinsic) • Hairpin loop formation • Inverted repeats in DNA • Usually GC rich regions (3-H bonds) • Rho-dependent • Rho-protein travels along RNA transcript until it reaches RNA polymerase • Causes RNA polymerase to dissociate from DNA • Rho protein: ATPase with helicase activity • Separates RNA-DNA hybrid helix releasing mRNA Core enzyme - NO Sigma subunit Holoenzyme - Sigma subunit: Enables RNA polymerase to recognize promotor regions on DNA RNA Polymerase (Prokaryotes) • Moves from 3’ to 5’ on DNA • Synthesizes mRNA from 5’ to 3’ • Function • Recognizes promotor region (nucleotide sequence) • Does not require a primer • Unwinds DNA helix (about 14 bp) • Makes a complementary RNA copy of the DNA template strand • Recognizes the termination region • Has exonuclease activity • Synthesizes all RNA except for RNA primers (synthesized by primase) • Has no proofreading activity • After polymerase has bound tightly to DNA and elongation begins, sigma subunit is released as polymerase proceeds beyond the promoter • Substrate: ribose nucleoside triphosphates • releases phosphate each time a nucleoside monophosphate is added to a chain • Moves from 3’ to 5’ on DNA template strand • Synthesizes mRNA from 5’ to 3’ • DNA non-template coding strand = RNA transcript (T instead of U) • mRNA is complementary to DNA template (anti-sense) strand and identical to coding (sense) strand (except U for T) • RNA primers are not transcribed Eukaryotic RECIPE • Enzyme TRANSCRIPTION: Eukaryotic genes • RNA Polymerase II • DNA Topoisomerase Initiation Inhibited by Alpha- • Promoter amanitin • CCAT box • RNA Polymerase II binds to promotor • TATA box • General transcription factors facilitate recognition + binding • Transcription factors CAAT box + TATA box • General • Specific Elongation • Enhancers • Formation of transcription bubble – local unwinding of short stretch of DNA • Generation of supercoils DNA topoisomerases Termination Transcription Factors • Initiation of eukaryotic transcription requires General TF • Help RNA polymerase II recognize promoters • Bind to promotors on DNA • Promotors: CAAT box + TATA box • Do not necessarily bind directly to promoters • • General transcription factors • Specific Transcription factors • Modulate the frequency of initiation • Mediate the response to signals such as hormones • Regulate which genes are expressed at a given point in time • TF II: A, B, D, E, F, H (not C, G) • A - helps stabilize binding TF II D • B - interacts with TBP, helps recruitment of RNA Pol II • D (TBP) - binds TATA promoter. Recruits TF II B • E - binds pre-initiation complex • F - interacts with TBP and helps RNA pol II bind promoter. Prevents RNA Pol II to bind other DNA • H - has ATPase activity (helicase) and kinase activity (elongation) Transcription Factors TBP = TATA-binding protein RNA Polymerase (Eukarytoes) RNA Polymerase I rRNA RNA Polymerase II mRNA * RNA Polymerase III tRNA (+other small) • RNA Polymerase II does not recognize and bind promoters Enhancers • Increase the rate of initiation of transcription by RNA polymerase II • Actual DNA Sequences (part of gene) • Located • upstream or downstream of the transcription start site • close to or thousands of base pairs away from the promoter • occur on either strands of DNA • Bind specific TFs: Functions by binding proteins which themselves bind RNA polymerase II Post-transcriptional modifications of mRNA Post-transcriptional modifications YouTube • 5’ capping • 7-methylguanosine attached to 5’ end of mRNA • Enzyme involved: RNA triphosphatase, guanylyl transferase, methyl transferase • Poly-A tail • Up to 200 residues • Enzyme involved: poly-A polymerase • Removal of introns by splicing • Introns = intervening sequences • snRNA (snurps) = facilitate removal of introns • Eukaryotes: mRNA – monocistronic: information from just 1 gene • Prokaryotes: mRNA - polycistronic Post-transcriptional Modification: SPLICING 1. Spliceosome-mediated 2. Self-splicing Watch this: https://www.youtube.com/wa tch?v=0BOWnqRVLHU Inhibitors of Transcription Molecule Inhibits Alpha-amanitin Eukaryotic RNA Polymerase II Rifampicin Prokaryotic RNA Polymerase Actinomycin D Inhibiting DNA, preventing elongation TRANSLATION TRANSLATION Location • Prokaryotes - cytoplasm • Eukaryotes - cytoplasm TRANSLATION Characteristics of Genetic Code • Specificity/unambiguous • A given triplet specifies exactly one amino acid – the same amino acid • Knowing sequence of nucleotides, we know sequence of amino acids • Universality • Highly degenerate • A given amino acid may have more than one triplet coding for it • Arginine: 6 different codons • Met + Trp: just 1 coding triplet • Non-overlapping • Code is read from a fixed starting point as a continuous sequence of bases • No punctuation • Nothing that separates codons TRANSLATION tRNA • RNA • Structure • Anticodon • 5’ end • 3’ end: Site of a.a. attachment (CCA) • Ester linkage • Charged (Activated) tRNA • a.a. attached • Codon determines a.a. on tRNA • Wobble Hypothesis • Allows some tRNAs to recognize more than one codon for a specific a.a. Wobble Hypothesis • Degenerate genetic code • 61 codons, 48 tRNA TRANSLATION Ribosomes • Protein synthesizing molecules • Made up of: protein + rRNA • Small subunit + Large subunit • Prokaryotes: 30S + 50S = 70S • Eukaryotes: 40S + 60S = 80S • Sites A (Aminoacyl-tRNA) Binds incoming aminoacyl-tRNA P (Peptidyl-tRNA) Codon is occupied by peptidyl-tRNA E (Exit) Occupied by empty tRNA TRANSLATION Translation video STEPS OF TRANSLATION: Initiation, Elongation, Termination INITIATION 1. Small subunit of ribosome binds to specific site on mRNA and recognizes start codon: AUG • Prokaryotes: Shine-Delgarno Sequence (towards 5’ end) • Eukaryotes: 5’-cap (5’ end) • Small subunit moves down scanning until encountering AUG 2. Initiator tRNA recognizing AUG: start codon • Facilitated by Initiation Factors (IF) eIF-2-GTP • Prokaryotes: IF-2-GTP • Eukaryotes: eIF-2-GTP : regulator of Iniitation • Initiator tRNA enters P site on small subunit • Prokaryotes: initiator tRNA carries formyl-methionine • Eukaryotes: Initiator tRNA carries methionine 3. Large subunit joins complex • GTP on (e)IF-2 is hydrolyzed to GDP Differences between Eukaryotes and Prokaryotes in Translation Eukaryotes Prokaryotes Binding of mRNA to - Cap at 5’ end of mRNA binds eIFs Shine-Dalgarno sequence small ribosomal subunit and 40S ribosomal subunit upstream to initiating AUG containing tRNA(Met) binds to complementary - mRNA is scanned for AUG start sequence in 16S rRNA codon (ribosomal subunit) First amino acid methionine Formyl-methionine Initiation factors eIFs (12 or more) IFs (3) Ribosomes 80 S (40 S + 60S subunits) 70S (30S + 50S subunits) STEPS OF TRANSLATION: Initiation, Elongation, Termination ELONGATION = addition of a.a to carboxyl end of growing chain Ribosome moves from 5’-end to 3’-end of mRNA 1. tRNA recognizes next codon, carrying 2nd amino acid and moves into A-site 2. (Formyl-)methionine carried by tRNA in P-site is joined to 2nd amino acid (in A- site) by peptide