Lecture 24: The Genetic Code

Interconvert between DNA, mRNA, and polypeptide sequences mRNA, tRNA, aaRS enzyme roles Anticodons and 3 stem How is the genetic code embedded in an aaRS enzyme? Wobble at the level of base-pairing Why are adenosine demaminase enzymes essential for synthdesis of wobble tRNAs? Biochemistry of aaRS enzymes How do some aaRSs proofread, and why
Protein synthesis is a BIG DEAL o Most abundant macromolecules in the cell o Vast majority of enzymes are proteins o 90% of a cells energy can be expended making proteins o 25% of E.colis dry mass can be ribosomes; almost 1,000 new ribosomes made per E.coli per minute o Many key antibiotics target protein synthesis Template-mediated flow of biological sequence information o Transcription and translation facilitate regulation and amplification in gene expression Regulate at both levels o One gene many mRNA millions of proteins o Can make many mRNA transcripts, and thus many proteins, or only a few transcripts

Genetic Code= set of RULES by which a linear sequence of nucleotides specifies the linear sequence of a polypeptide o What allows us to interconvert between nucleotides and polypeptides Discovery of the genetic code o Early on, could not sequence DNA and had only rudimentary means of sequencing proteins o First key question: how nucleotides needed to code for amino acid? Crick and Brenner found that code was in units of 3 nucleotides: using mutant bacteriophage strains with inserted or deleted based pairs, found that mutants with three nucleotides inserted or deleted, not two or four, could be made functional again

Non-overlapping code No spacers between codes (no punctuation marks Used the process of elimination to settle on this model Because there are 64 possible codes (4 nucleotides), the code must be degenerate Reading along DNA or RNA strand in one direction, there are three possible reading frames In dsDNA, there are 6 possible reading frames: 3 on top and 3 on bottom A +2 change in reading frame is equivalent to moving the frame -1 How do we know where to start reading?

Which codons specify which amino acids? Once we understand syntax (how to read), we need to understand vocabulary Decoding: First, found that the synthesis of long tracts of poly-U resulted in chains of Phe (this, UUU Phe) Used synthetic RNA with A and residues in a 5:1 ratio and then determined identity and quantity of incorporated amino acids o Gave AA tentative assignments of nucleotide composition (not sequence) o Could guess frequency of incorporation based on nucleotide composition and compare this to observed frequency Later on, did binding assays and found which tRNA were in the binding site Codon assignment: Genetic code table o Some AA have multiple codons coding for them o In most cases , AUG (Met) is the start, of the figurative capital letter UAA, UAG, UGA are stop codons, or periods U is normally in the middle of hydrophobic residues so that errors result in appropriate substitutions that are less likely to impair protein function (Leu, Ile, Val) How is the genetic code implemented? o

Brenner-Crick adaptor hypothesis in On Degenerate Templates and the

Adaptor Hypothesis they had no idea what the actual parts were, but they had ruled out other ideas AAs combine chemically, at special enzyme, with small molecule which, having specific H-bonding surface, would combine specifically with the nucleic acid template (enzyme places orrect AA on correct adaptor) 20 adaptor molecules, one for each AA, and 20 difnt enzymes to join AAs to adaptors

Secondary and tertiary tRNA structure: The adaptor is tRNA (transfer RNA)
o Anticodon that base pairs with a specific codon o Base pairing occurs when chains run in opposite directions (tRNA runs in direction opposite of mRNA) o Always an A at the end of the amino acid arm

o anticodon

general shape of most tRNAs is the same Most dramatic variation at the

There are some internal differences Base pairing occurs between D arm and T arm internally and between the two arms: this bending is what provides stability

Secondary structure: double helices similar to A-DNA A is the more dehydrated form of DNA; even fully hydrated RNA is in this form A form has more tilt and a more open conformation (more slide and roll)

Wobble o Could have 64 different tRNAs, but 40 is more typical o tRNAs need to be able to interact with more than one codon; in third position, some tRnAs can form more than one contact o wobbling allows us to be more efficient: errors only 39/40 times as opposed to 63/64 times o example:

 Alanine tRNA recognizes GC(A/C/U) codons anticodon has I-G-C (5-3), with inosine in the wobble position o one codon recognized: C (G) and A (U) In right context, C o o can pair with more than one case Two codons: U (A/G), G (C/U) Three codons: I (A/U/C) Inosine created by adenosine deaminase enzyme recognizes wobble base and deaminates Inosine: inosine base is hypoxanthine

tRNA synthetases assign the codons o must recognize correct tRNA and then conjugate AA o 20 kinds to join 20 different AA to d20 different adaptors o There are classes of aminoacyl-tRNA sdynthetases based on type of amino acid created

1. Activation of amino acid releases pyrophosphate Then PPi 2Pi and heat via pyrophosphatase Mized anhydride is tightly bound to active site

2. Formation of charged tRNA when amino adenylate is joined to tRNA aminoacyl tRNA is in the activated state differences in where the hydroxyl attacks by end, everyone ends up on 3!

Lecture 25: Protein Synthesis explain main structural features of ribosomes and know (roughly) how many DNA and protein subunits they contain Main functions big subunit Main functions small subunit What is a polysome? How ribosomes place themselves on the start codon; different in bacteria and eukaryotes. o Compare and contrast these mechanisms Understand what bacterial IF-1 and IF-3 do Understand what eukaryotic eIF4 complex does Understand what a polycistronic mRNA is. Why are these very common in bacteria and rare in eukaryotes? Why is coupled transcription and translation in bacteria but not ukaryotes fMets role in bacterial inititiation

Template-mediated polymer synthesis has 3 stages

With a few exceptions, ribosomes are responsible for all protein synthesis 1. Initiation: locate the starting point on the template; assemble the polymerization machinery An error of +/- 1 will lead to a non-functional protein Thus, initiation must be very precise o 2. Elongation: add a promoter to the growing polymer, as specified by the sequence on the template; repeat o 3. Termination: cease elongation disassemble the elongation hardware Take apart and recycle Polypeptide must be severed from ribosome Translation parts: o mRNA = template o 20 amino acids o About 40 tRNAs o 20 aminoacyl tRNA synthases o ATP and GTP Energy sources for things like moving the template o Ribosome Small subunit = decoding center Large subunit = peptidyl transferase center o Inititation factors o Elongation factors o Termination factors WHAT YOU NEED TO KNOW ABOUT RIBOSOMES AND PROTEIN SYTHESIS: o o

Size of o o o o

ribosome Erythrocyte: 8,000 nm Bacterium: 2,000 nm DNA: 2 nm wide. 300 bp are 100 nm long Ribosome: 25 nm BIG o Some tRNAs are as large as enzymes Composition of ribosomes: o Nobel prize won in 2009 for studies of structure and function of the ribosome o Ribosomal proteins lie mainly on the surface o Proteins are mostly add-ons o The catalytic site itself is ribosomal o The folding of ribosomal subunits is highly conserved between eukaryotes and bacteria Eukaryotic has more stuff and is larger, but in general the structures carrying our core activities are identical There is both evolutionary conservation and divergence of ribosomal proteins o With regard to small subunit: some cores are found in all kindgddomes; some cores are only archaeal; some proteins or extensions are uniquely eukaryotic

Distinctive features of the eukaryotic ribosome map to the cyoplasmic surface o There are both eukayote-specific RNA loops and proteins o Conserved region surrounding the polypeptide exit tunnel

Chaperones and docking proteins have to interace with the ribosome there

Polysome: mRNa with multiple translating ribosomes o At the 5 end, the polypeptides are smaller because the ribosome has not moved as far down the chain

Translation o 1. Inititation: risibome placed on the start codon o 2. Elongation: mRNA-templated polypeptide polymerization o 3. Termination: polypeptide and mRNA are released To get out of mucleus, mRNA must be capped, tailed, and spliced o Cartoon below applies to bactiera; direction is same in eukaryotes but transcription is in nucleus and translation is in cytoplasm

In bacterial initiation, ribosome small subunit binds directly to Shine-Delgarno initiation sites on the mRNA oCapping only occurs in eukaryotes oProteins created below are often functionally related oCan control stoichiometry of protein subunits because different ribosome binding sites have different efficiencies oSequence in binding sites form base pairs within the ribosomes structural RNA

End of rRNA and sequence pair together

In eukaryotic inititation, the small subunit binds the 7-methyl-G-cap, then scans 5 3 to find a start codon oEukaryotes need a 5 cap for translation to occur oMotors along using an ATP-dependent motor oEukaryotes rarely have regions that code for more than one polypeptide Polymerase has to fall off at the end

Lecture 25: The Ribosome and Protein Synthesis II the N-terminus of the growing polypeptide worms through an exit tunnel in the large subunit oRegion around the tunnel is highly conserve oFolding is cotranslational Elongation cycle: the hybrid state model of elongation

oEf-G is a motor protein that hydrolyzes GTP and causes a conformational change that forces the A/P tRNA to fully occupy the P site, the P/E tRNA to fully occupy the E site, and the mRNA to shift 3 nucleotides down relative to the ribosome due to its association with these tRNA molecules 1. aa-tRNA selection and accommodation o tRNA enters in combination with elongation factor two (Ef-Tu) = aa-tRNA: Ef-Tu: GTP oThis combination adopts a bent conformation in the decoding site the base pairing must be strong to keep the tRNA in the A site, ensuring that the right tRNA has entered o The aminoacyl group attached to acceptor stem varies o Ef-Tu promotes GTPdependent translocation of nascent protein chain from the A-site to the P-site of the ribosome oBefore we move to peptidyl transfer, there are still two chances for an incorrect aa-tRNA to fall off a true proof reading mechanism 1: incorrectly base-paired tRNAs dissociate before GTP hydrolysis (recognition stage)

 2: dissociate after GTP hydrolysis (before accommodation stage) energy purely used for proofreading Tetracycline binds the 30S subunits A-site, blocking entry of the aatRNA-Ef-Tu complex

kinetic proofreading: the timer function of Ef-Tu is critical for accuracy

a slowly-hydrolyzing GTP analog increases the accuracy of translation (more time for dissociation to occur) at the expense of speed; the

misincorporation rate goes down at the cost of efficiency

2: PEPTIDYL TRANSFER Process only occurs if accommodation happens and relies on ribosomal catalysis Note: N-formyl group occurs on bacteria only Puromycin is a chain terminator that binds in PTC and mimics aminoacyl-tRNA o The chain cannot be transferred to another tRNA in the A site The fragmentation reaction mimics peptidyl transferase, using oligonucleotide fragment as peptidyl-tRNA analog and puromycin o The activity of peptidyl transferase is measure by formation of the product shown below o peptidyl transferase is RNA, NOT protein Noller: used every type of extraction to get rid of proteins and the chemistry still happened know that it was peptidyl transferase because activity inhibited by peptidyl transferase- specific antibiotics thus: the ribosome is a ribozyme o there are no proteins within 18 A of peptidyltranster active site peptidyltransferase center (PTC) is within large subunit many antibiotics bind in the PTC (including puromycin) o chloramphenicol: inhibits PT rxn and blocks exit tunnel o Macrolide antibiotics plug the exit tunnel from the inside, preventing polypeptide escape o

3: TRANSLOCATION AND TH Process powered by Ef-G, a GTP-p ohydrolyzes GTP and causes a c forces the A/P tRNA to fully occu tRNA to fully occupy the E site, a nucleotides down relative to the association with these tRNA mol oEf-G:GTP (used for tanslocation conformation to aa-tRNA:Ef-Tu:G decoding) because it also binds is entirely protein subunits rotate with respect to one

4: TERMINATION The polypepetidde chain is covalently attached to a tRNA in the P site and threaded through the large subunit Release factor enters the A site and triggers release o Also shaped like tRNA! o Made of protein o Ex: GGQ and SPF are parts of release factor that are thought to enter and catalyze hydrolysis reaction The ribosome moves the polypeptide to H2O so that the link between the polypeptide and tRNA is hydrolyzed

Ef-G cooperates with release factors to disassemble spent tRNAs, subunits, mRNA 0. mRNA is being threaded through, with elongated polypeptide moving out. Termination codon is hit. 1. Release factor 1 or 2 enters 2. Hydrolysis occurs and protein released 3. Release factor 3, a GTPase, enters and causes the dissociation of release factors 1, 2, 3, and the deacylated tRNA 4. Now, nothing in the A site and ribosome recycling factor can enter 5. Ef-G and ribosome recylcing factor bind and the two work as a crowbar to cause subunit dissociation