May 24, 2006Fig 32-6: Genetic Map of bacteriophage x174 as determined by DNA sequence analysis.

Table 32-2: The Standard Genetic Code (do not need to memorize)o All codons have meaningo UAA, UAG, UGA do not code for an amino acid and are called “nonsense” codons, they serve as

termination sequenceso The genetic code is unambiguous, each of the 61 “sense” codons encodes for only 1 AAo The genetic code is degenerate. Except for Met and Trp each aa is encoded by more then one codon.

These are called synonymous codons.o The genetic code is nearly universalo 1st position on lefto 2nd is horizontalo 3rd is on righto start does for Met

Codons for the same (or similar) amino acids tend to be similar in sequence:o XYX likely encodes for a hydrophobic aao XRX likely encodes for a polar or charged aao GAX encodes for the acidic aao Y is a pyrimidineo R is a purine

Third base degeneracy—(5` residue of the anticodon)o Often the 3rd base in a codon is irrelevanto Eight aa are encoded by the 1st two bases:

o Leu: CUX Pro: CCX Arg: CGX Val: GUX Thr: ACX Gly: GGX Ser: UCX Ala: GCX

o Seven aa are encoded by a pyrimidine base in the 3rd positiono Phe: UUY Asn: AAY Ser: AGY Tyr: UAY Asp: GAY His: CAY

Cys: UGYo Five aa and the stop codon are coded by a purine base in the 3rd position:

o Leu: UUR Glu: GAR Gln: CAR Arg: AGR Lys: AAR stop: UARo One aa is coded by any base but G in the 3rd position

o Ile: AUX x=U, C, or Ao Two aa are coded where the 3rd base must be a G:

-have overlapping genes-gene for B entirely embedded in gene for A, just have different reading frames-complex genome-doesn’t occur in eukaryotes, only phages w/ ssDNA

o Met: AUG Trp: UGGo The stop codon is coded w/ the requirement that the 3rd base must be an A

o Stop: UGA; unlike the other stop codons (UAR) this stop codon is sometimes used to code for selenocysteine, Sec, aka the 21st aa

In 1966, Francis Crick proposed the “Wobble hypothesis.”—the 5` base in the anticodon loop is capable of “wobbling” during translation, allowing it to make alternative, non-Watson-Crick H-bonds w/ different codon bases5` anticodon base pairs w/ 3` codon baseC GA UU A or GG U or CI U, C or A

1st base in codon pairs w/ 3rd base in anticodon

ribosome forms peptide bond between amino acids

Fig 32-11: Structure of Yeast tRNA

Fig 32-12: Tertiary base pairing in yeast tRNAo Typical of long range bpso Interactions stabilize hammer or T-like structure

How is the right amino acid matched to its corresponding tRNA?o This is rather important as once an amino-acyl-tRNA is synthesized, the amino acid becomes a “passive

participant” in translationo Aminoacyl-tRNA synthetase

o Cells have 20 different aminoacyl-tRNA synthetases, one for each aa (except Lys, which in E.coli has 2)

o The formation of aminoacyl-tRNAs occurs in 2 steps:1. the aa is accepted by the synthetase and adenylated2. the proper tRNA is accepted by the synthetase and the aa is transferred to the 3` terminal

residue of the tRNAOverall rxn:

There are 2 distinct classes of aminoacyl-tRNA synthetases: Class I nad IIo Class I: monomer, first adds the aa to the 2`-OH of the terminal adenylate residue of the tRNA before

shifting it to the 3`-OH. Class I enzymes bind to the tRNA acceptor stem helix from the minor groove site

o Class II: dimer/tetramer, adds the aa directly to the 3`-OH of the terminal adenylate residue of the tRNA. Class II enzymes bind to the tRNA acceptor stem helix from the major groove site.

Common Features of tRNAo 5` terminal phosphate groupo acceptor stem (7 bp, often w/ non WC bp)o D loop (3-4 bp stem, often w/ dihydrouridine, D, in the

loop)o Anticodon loop (5 bp stem)o TψC loop (5 bp stem w/ a loop containing pseudouridine,

ψ)o Terminal CCA sequence w/ a free 3` OH o The tertiary structure is maintained by H-bonds and

stacking interactionso Clover leaf structureo Generally 76 nt in lengtho Variable loop is most variable part of tRNAo 3-D shape stays pretty much the same

How does a synthetase recognize its cognate tRNAs?The recognition occurs via various elements, which include one or more of:

o At least one base in the anticodono One or more of the 3 bps in the acceptor stemo The base at position 73 (unpaired base preceding the CCA end)

Minor groove

Major groove

Class I Class IIArg AlaCys AspGln AsnGlu GlyIle HisLeu LysMet PheTrp ProTyr SerVal Thr

For example:E.coli glutaminyl-tRNAGln synthetase uses:

o The central U in the CUG anticodono Contacts from the carboxyl grp of Asp235 to the 2-NH2 grp of G3 (minor groove) of the G3:C70 bp in the

acceptor stemo Contacts w/ the “discriminator base”o A mutant synthetase (D235N) will acylate noncognate tRNAs w/ Gln

May 26, 2006

Recognition of the Anticodono Changing the anticodon of either tRNATrp (CCA) or tRNAVal (CAC) to CAU, the anticodon for the

Metionine codon AUG, transforms each of the tRNAs into a substrate for methionyl-tRNAMet synthetaseo Reversing the Met anticodon of tRNAMet (CAU) to UAC transforms it into a substrate for valy-tRNAVal

synthetaseo Red dots are being recognized by synthetaseo Anticodon CAU finds codon AUGo If you change the anticodon, it has a large effect of what gets put onto tRNA

A single acceptor stem bp defines tRNAAla

o All tRNAAla, from archaebacteria to eukaryotes, possess the noncanonical G3:U70 bp in the acceptor stem. Changing this to G:C, A:U, or U:G abolishes its ability to be aminoacylated w/ Ala (when base paired w/ Uracil, the 2-NH2 of G3 in the minor groove lacks an H-bonding partner

Mutation of a sense codon to a nonsense codon (UAA, UAG, or UGA) results in a truncated polypeptide.However, mutations elsewhere in the genome can suppress the effect of nonsense mutations (nonsense suppression). These suppressions arise from mutations in the tRNA genes that alter the anticodon so that the mutant tRNA read a stop codon and insert an amino acid. For example, changing the anticodon of tRNATyr from GUA to CUA allows tRNA to read the UAG stop codon and insert a Tyr. Suppression tRNAs are generated from minor tRNA species w/in a set of isoacceptor tRNAs, so there is no loss of essential tRNA.UAG: amber stop codon UAA: ochre stop codon UGA: opal stop codon

Ribosomeso Ribosomes are large, compact ribonucleoprotein complexes that are found in the cytosols of all cells

(also in the mitochondrial matrix and the stroma of chloroplasts). o They are composed of two subunits, each of which contains ribosomal RNAs (rRNAs) and proteins.o In prokaryotes, ribosomes have a sedimentation coefficient of 705 and the subunits are a 305 particle

and a 505 particle

o In eukaryotes, the ribosomes have a sedimentation coefficient of 805 and the subunits are a 405 particle and a 605 particle

In prokaryotic ribosomes, o there is only one copy of each ribosomal protein per 705 ribosome, except for protein L7/L12, of which

there are 4 copies. o Only one protein is common to both the large and small subunits: S20=L26. o The largest ribosomal protein is S1 (557 aa, 61.2 kDa) and the smallest is L34 (46 aa, 5.4kDa). o There is very little sequence homology between ribosomal proteins, although they tend to be rich in Lys

and Arg, and have very few aromatic aa residues.o However, comparing ribosomal proteins between different organisms reveals considerable evolutionary

conservation of sequence

30S + 50 S 70 SThe ribosome has 3 sites for RNA binding:

1. A site (aminoacyl)2. P site (peptidyl)3. E site (exit)

Mechanics of TraslationThere are 3 steps in translation:

1. Initiation2. elongation 3. termination

Each of these stages requires specific proteins to interact w/ the mRNA, tRNA and/or the ribosome. These proteins are the initiation factors (IFs), the elongation facts (EFs) and the release factors (RFs).

Initiation: In prokaryotes, the first aa is formylmethionine

The same aminoacyl-tRNA synthetase attaches methionine to both tRNAMet and tRNAFMet. However, Met-tRNAMet is not a substrate for formylation by transformylase.fMet-tRNAfMet is recognized by IF-2 whereas Met-tRNAMet is recognized by EF-Tu.E.coli proteins are postranslationally modified by deformylation of the fMet residue.