Alberts Johnson Lewis Morgan Raff Roberts Walter Molecular Biology of the Cell Sixth Edition Chapter 6 (pp. 333-368) How Cells Read the Genome: From DNA to Protein Copyright Garland Science 2015 Genetic Information Flow in the Cell DNA (Information stored on hard disk) Transcription Precursor messenger RNA RNA Processing Nucleus Cytoplasm Mature mrna Protein (Final output) Translation
Control of Gene Expression Many steps between transcription and functional protein All reactions can (potentially) be regulated Mature Eukaryotic mrnas Are Selectively Exported from the Nucleus
Prok vs Euk: Differences in Translation Polycistronic Monocistronic Prok vs Euk: Differences in Translation
Eukaryotic Protein Synthesis Translation of mrna mrnas: the good guys and the bad guys 5 cap AUG context Kozak Consensus= GCC(A/G)CCAUGG 5 UTR length 5 UTR 2 o structure Upstream AUG 3 poly(a) tail Initiation the rate-limiting step Elongation Termination trna Molecules Match Amino Acids to Codons in mrna
trna Molecules Match Amino Acids to Codons in mrna Specific Enzymes Couple Each Amino Acid to Its Appropriate trna Molecule Aminoacyl trna synthetases
Specific Enzymes Couple Each Amino Acid to Its Appropriate trna Molecule Eukaryotic Ribosomes 40S Small Subunit 18S rrna Transcribed by RNA Pol I 60S Large Subunit 25S rrna 5.8S rrna 5S rrna I I III
The Nucleolus Is a Ribosome-Producing Factory The Nucleolus Is a Ribosome-Producing Factory
The Ribosome Is a Ribozyme The RNA Message Is Decoded in Ribosomes
[Mg 2+ ] = 1 2 mm, 80S ribosomes predominant Mechanism for ribosomal dissociation unclear. eif3 ( 8 subunits) : binds to 40S subunit eif6: binds to 60S subunit (??) Active dissociation??? Formation of stable ternary complex (3 o ) Met-tRNA i eif2 (α/β/γ subunits) GTP
Unwinding 2 o Structures in 5 UTR By eif4f: the RNA Helicase Complex eif4e: cap-binding protein; 0.2 copies/ribosome eif4g: the platform, binds to eif3/eif4a/eif4e/pab1p eif4a: DEAD-Box RNA helicase eif4b: RNA-binding protein 48S complex Ribosome Scanning Model: Locating the 1st AUG assisted by eif1 and eif1a
eif2b: the recycler Promote GDP/GTP exchange eif5: the fidelity factor Assess met-trnai/aug interact. Promote GTP hydrolysis by eif2 Release other translation factors eif5b
Basic Aspects of Translation trnas stretches across both subunits A site (Acceptor) presents the codon for next amino acid; binds incoming aminoacyl-trna P site (Donor) represents the most recently added aa; occupied by peptidyltrna carrying the nascent polypeptide Footprint of ribosome is ~35 bases of mrna Elongation: Placing the 2nd charged trna to the A site eef1a/trna/gtp The proofreading step: Checking trna/anticodon Interaction at A site
Peptide bond formation Peptidyltransferase = RNAs or Proteins?
Polysome: a unit consisting of an mrna associated with several ribosomes. Typical unit is 8 ribosomes per eukaryotic mrna Polysome Profile Analysis 48S 80S couple [60S] Halfmer 80S 1.5mer Free 40S Free 60S [60S] Halfmer Dimer 2.5mer Trimer
Wild-type dbp3 mutant Proteins Are Made on Polyribosomes
Cap + Poly(A) - mrna Initiation eif4g as an assembly platform eif4e (N-term.) eif3 (Mid.) eif4a (C term.) Hentze, M (1997) eif4g: a multipurpose ribosome adapter. Science 275, 550. Cap - Poly(A) + mrna Initiation eif4g as an assembly platform: Pab1p (Mid.) eif3 (Mid.) eif4a (C term.)
Cap - Poly(A) - (viral) mrna Initiation Inhibits host translation: cleaving the N-term. eif4g Use truncated eif4g to bind to IRES on viral mrna Recruit 40S/eIF3 complex to IRES for translation Cap + Poly(A) + mrna Initiation Synergy between 5 & 3 ends
Figure 8.41 Splicing mechanisms in group I and group II self-splicing introns and spliceosomecatalyzed splicing of pre-mrna. Which Came First? DNA, RNA, or Protein? Information & Enzymatic Activity RNA Rules! RNA World! Examples: Self-splicing catalytic RNA, Ribosome
Introduction All Present-Day Cells Use DNA as Their Hereditary Material
RNA Continues to Play a Central Role in Modern Cellular Machinery Active center of ribosome is entirely made of RNAs
Spliceosome Structures Solved: >35 Proteins and 4 snrnas 11 September 2015 Shi Yigong Kiyoshi Nagai Reinhard Luhrmann 23 September 2016
1993 Nobel Laureate