Chapter-9-for-bb-lecture-notes-9.pdf

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Chapter 9 for BB - Lecture notes 9 Mendelian And Molecular Genetics (University of Illinois-Chicago)

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Chapter 9 •  Protein structure •  The genetic code •  Translation

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•  Protein structure •  Genetic code •  Translation

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Proteins are polymers (poly = many; mer = part) made of amino acids attached end to end in a linear string. The general formula for an amino acid is: H2N CHR COOH in which the side chain, R, can be anything from a hydrogen atom to a complex ring structure.

NH2-CH-COOH R

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There are 20 common amino acids found in terrestrial life forms, each with unique chemical properties that are determined by the R groups.

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Amino acids are connected by peptide bonds between the carboxyl end of one and the amino end of the next, thus forming a linear polypeptide chain.

Directionality!

Carboxyl end

Amino end Downloaded by Soji Adimula ([email protected])

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Proteins have four levels of structure •  Primary (1˚) structure refers to the linear sequence of amino acids in a polypeptide •  Secondary (2˚) structure refers to interactions between amino acids that are close together. 2˚ structure is usually due to hydrogen bonding between the CO and NH groups of nearby amino acid residues (e.g., α-helix and β-pleated sheet). •  Tertiary (3˚) structure refers to the 3D architecture of a protein. It is determined by electrostatic, hydrogen, and Van Der Waals bonds between R groups (often distant). •  Quaternary (4˚) structure refers to the binding together of two or more individual polypeptides to form a multimeric protein (e.g., hemoglobin).

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Primary (1˚) structure — the sequence of amino acids in a chain

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Secondary (2˚) structure — the first level of 3D folding; determined by H-bonding between nearby NH and CO groups The two most common types of secondary structure: α-helix and β-pleated sheet

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Tertiary (3˚) structure — the folding of the secondary structures to form the final 3D shape of a polypeptide

The β chain of hemoglobin

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Some proteins possess Quaternary (4˚) structure — several polypeptides join together to form a multi-subunit structure

Hemoglobin is made up of two α and two β chains.

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Protein structure summary Ø  Primary structure (the sequence of amino acids) determines the secondary and tertiary structures of the protein (the way the string of amino acids folds in 3D space). Ø  The amino acid side chains determine the folding of the protein, and provide functionality to the interaction surfaces and to the active sites of enzymes.

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•  Protein structure •  Genetic code •  Translation

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Demonstration of the colinearity between gene & protein In 1963, Charles Yanofsky demonstrated what was inferred from the structure of DNA: there is a correspondence between the linear sequence of a gene and its encoded polypeptide. TrpA

TrpA

Gene: Mutations were mapped on DNA by P1 transduction (recomb. analysis). Protein: The wild type and mutant proteins were sequenced. Conclusion: The linear sequence of nucleotides in a gene determines the linear sequence of amino acids in its protein.

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Overview of the genetic code •  The questions –  Is the code overlapping or nonoverlapping? –  How many letters (nucleotides) are there per word (amino acid)? –  What is the code and what are its properties?

•  The tools –  –  –  –  –  – 

Logic and insight DNA and protein sequencing Synthetic RNA and programmed synthesis of polypeptides Powerful genetic systems such as the rII locus of phage T4 tRNA conversion mini mRNAs

•  The answers –  –  –  – 

The code is nonoverlapping and each codon has three letters. All but three codons specify an amino acid; three codons specify stop. The amino acids are illiterate The code is read in a polarized fashion and is degenerate

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Is the genetic code overlapping or nonoverlapping? reading 3 nts at a time

also reading 3 nts at a time

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How can we determine if the genetic code is overlapping or nonoverlapping? Experiment: Sequence mutant genes and their protein products. Ø  If the code is nonoverlapping, then a single base change should alter only one amino acid in the protein. Ø  If the code is overlapping, then a single base change should alter > 1 amino acid in the protein (e.g., if 2 bases overlap, a single base change will alter 3 adjacent amino acids).

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Overlapping or nonoverlapping? Result: Mutation of a single base in a given gene results in one amino acid change in corresponding protein. Conclusion: The genetic code is nonoverlapping ! This is true for a given protein. It is possible, although rare, for two different proteins to be encoded by a single mRNA sequence read in two different frames.

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How many letters per word in the genetic code? Logic: There must be at least 20 words (codons) to encode all 20 amino acids. -  1 is not enough – only 4 codons (A, T, G, C) -  2 is not enough – only 4 x 4 = 42 = 16 codons -  3 is more than enough – 4 x 4 x 4 = 43 = 64! Therefore, there must be at least 3 nts per codon.

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But is it 3 or is it more than 3? In 1961, Francis Crick and Sidney Brenner demonstrated that the genetic code uses three letters (nucleotides) per word (amino acid). Ø  Crick et al. used the rII locus of the T4 phage. Ø  They began by mutagenizing the phage with proflavin, which induces single base pair insertions and deletions, and recovering rII mutants. What is the phenotype of rII mutants?

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Crick’s and Brenner’s experiment Goal: Isolate suppressors of mutations in the rII locus of T4 1. Induce rII mutations by proflavin, a mutagen that adds or deletes single base pairs in DNA. 2. Mutagenize the rII mutants resulting from step 1 using the same mutagen, and select for revertants that can grow on E. coli K. Wild type Growth on E. coli K: +

rII mutant -

Revertant +

Revertants can be: true revertants or suppressors

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True Revertant Example: Code:

ACT ACT ACT ACT ACT ACT

1st mutation: insertion of G

ACT GAC TAC TAC TAC TAC

True Revertant: Deletion of the same G

ACT ACT ACT ACT ACT ACT

Mutation

wrong codons Very rare

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Suppressor: a second mutation that counteracts the effects of the first mutation. By definition, a suppressor mutation is at a distinct site, and can be either intragenic or extragenic. Example: Code: ACT ACT ACT ACT ACT ACT 1st mutation: insertion of G

ACT GAC TAC TAC TAC TAC

2nd mutation: Deletion of C

ACT GAC TAT ACT ACT ACT

Mutation

wrong codons

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Experiment: Suppressors of mutations in rII locus of T4:

Growth on E. coli K:

Wild type +

rII mutant -

Revertant +

Q: How can you distingusish a true revertant from a suppressor? A: If the second mutation does not restore the wild type sequence, then the suppression of the mutant phenotype must be due to a mutation at a second site, and the 2 mutations can be separated by recombination with wild type. Downloaded by Soji Adimula ([email protected])

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Mut #1

Double mutant Mut #1 Suppressor

WT

2 single mutants

This is exactly what Crick and Brenner observed: second site mutations that suppressed the rII phenotype conferred by the first mutation, but which caused the same phenotype when isolated by recombination. Downloaded by Soji Adimula ([email protected])

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Crick’s and Brenner’s experiment continued •  In analyzing their data, Crick and Brenner found a striking pattern: v  Deletions (-) could suppress insertions (+). v  Insertions (+) could suppress deletions (-). v  However, changes of the same sign could not suppress one another,

with one important exception: triple mutations of the same sign (3 insertions or 3 deletions) often allowed wild type function!

•  Crick and Brenner concluded that the mRNA is read in a polarized fashion and proceeds three nucleotides at a time. Their observations can be explained as follows: –  Insertion and deletion mutations alter the reading frame, and thus are called frameshift mutations. –  Suppressor mutations introduce a compensatory change.

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Frameshift mutations The triplet code can be read in three possible frames

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Frameshift mutations Experiment: Suppressors of Mutations in rII locus of T4: Mechanism: The triplet code (3 nucleotides/amino acid) is read in a polarized fashion (i.e., in one direction), and can be read in three possible frames. First mutation: +/- out of reading frame, or frameshift mutation Second mutation: -/+ back to frame, and sequence is back to correct, back to sense In between: a short stretch of missense! (cannot be too long, or the protein will be nonfunctional) Downloaded by Soji Adimula ([email protected])

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Frameshift mutations Experiment: Suppressors of Mutations in rII locus of T4: Example: Code:

ACT ACT ACT ACT ACT ACT

1st mutation: insertion of G

frame-shift ACT GAC TAC TAC TAC TAC

2nd mutation: Deletion of C

back to frame ACT GAC TAT ACT ACT ACT

Mutation

missense codons Downloaded by Soji Adimula ([email protected])

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Why is the second mutation deleterious by itself? Experiment: Suppressors of Mutations in rII locus of T4: Example: Code:

ACT ACT ACT ACT ACT ACT

2nd mutation by itself: Deletion of C

frame-shift ACT ACT ATA CTA CTA CTA

Both the 1st and the 2nd mutations are frameshift mutations!! Mutation

missense codons Downloaded by Soji Adimula ([email protected])

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Observation: Triple mutations of the same sign (+/+/ + or -/-/-) can restore wild type function Example: Code:

ACT ACT ACT ACT ACT ACT….

3 deletions:

ACT ATC TCT ACT ACT….. Back to frame

Conclusion: the code is exactly 3 nucleotides per codon Mutation

missense codons

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The definitive evidence for polarized and framed reading of the genetic code George Steisinger used proflavin to induce mutations in the gene that encodes lysozyme, a protein with a known amino acid sequence. Wild type revertant

-thr-lys-ser-pro-leu-asn-ala-thr-lys-val-his-leu-met-ala-

Explain how this proves polarized and framed reading of the code. Question: Why do mutant alleles with intragenic suppressor mutations often encode proteins with only partial function? Question: How many reading frames are there for a given molecule of DNA? Downloaded by Soji Adimula ([email protected])

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A triplet code read in a single frame in a polarized fashion THEFATRATATETHEREDANTANDTHEBIGCOW THE FAT RAT ATE THE RED ANT AND THE BIG COW T HEF ATR ATA TET HER EDA NTA NDT HEB IGC OW TH EFA TRA TAT ETH ERE DAN TAN DTH EBI GCO W Crick could remove or or add one or What and can Brenner we deduce if we remove add one more bases in the rII gene and ask it or more letters in this sentence and whether ask still coded for amakes functional protein . whether it still sense ?

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How did Crick and Brenner infer that codons are 3 nucleotides long? THE X FAT RAT ATE THE RED ANT AND THE BIG COW H THF ATR ATA TET HER EDA NTA NDT HEB IGC OW THF ATR ATA HTE THE RED ANT AND THE BIG COW •  So how did they deduce that the code uses 3 letters per word? •  Why does a suppressor mutation cause loss of function when it is isolated? •  Why does a mutant gene with a downstream suppressor often only have partial function?

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Next property of the genetic code: degeneracy •  The genetic code is degenerate, which is another way of saying that it is redundant. This means that some amino acids are specified by more than one codon. •  Evidence for degeneracy of the code (Crick's supposition) –  Hypothesis: The code is not degenerate. Each of the 20 amino acids is specified by a single codon, and the other 44 codons specify no amino acid—i.e., they are nonsense (termination) codons). –  Predictions: •  All frameshift mutations should result in immediate (or almost immediate) termination of translation. Why? •  Suppression of frameshift mutations will not occur because translation will be terminated before a second site mutation can restore the reading frame.

–  Observations: •  Frame mutations usually do not result in immediate termination. •  Frameshift suppressors arise frequently.

–  Conclusion: The code is degenerate Downloaded by Soji Adimula ([email protected])

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Summary so far: The genetic code is nonoverlapping, read in a polarized fashion 3 nucleotides at a time, and is degenerate.

But what is the code?

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Cracking the Code #1: Random incorporation of nucleotides one at a time •  The first breakthrough was the production of simple, defined molecules of RNA. –  In 1961, Nirenberg and Matthaei succeeded in synthesizing RNA from scratch — i.e., in the absence of DNA — using the enzyme polynucleotide phosphorylase and randomly incorporated nucleotides.

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Cracking the Code #1: Random incorporation of nucleotides one at a time •  The first breakthrough was the production of simple, defined molecules of RNA. –  In 1961, Nirenberg and Matthaei succeeded in synthesizing RNA from scratch — i.e., in the absence of DNA — using the enzyme polynucleotide phosphorylase and randomly incorporated nucleotides. –  Why couldn’t they have used RNA polymerase?

•  Synthetic RNAs were used to program the synthesis of polypeptides in vitro using the translational machinery of E. coli reconstituted from purified components. •  The firt codon assignment was UUU. The polynucleotide -UUUUUUUUUUUUUUU- encodes polypeptide -Phe-Phe-Phe-Phe-. •  The AAA, GGG, and CCC codons can be assigned in the same way. Downloaded by Soji Adimula ([email protected])

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What is the code? The breakthrough was the ability to make RNA without DNA: Ribonucleotides (ATP, GTP, CTP, UTP) + the enzyme polynucleotide phosphorylase Single stranded RNA of random sequence (No need for transcription of DNA) This artificial RNA was then used to make the encoded polypeptide(s) in vitro Downloaded by Soji Adimula ([email protected])

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What is the code? Synthetic RNA: PNP + UTP (uracil triphosphate) UUUUUUUUUUU …, or Poly(U) Poly (U) + protein synthesizing machinery Protein: Poly-phenylalanine Phe-Phe-Phe-Phe-Phe-Phe … Nirenberg Downloaded by Soji Adimula ([email protected])

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What is the code? Conclusion: Codon UUU

aa phenylalanine

Later added: AAA, CCC, GGG CCC AAA GGG

proline lysine glycine

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Cracking the Code #2: Repeating nucleotide polymers •  In this approach, a repeating nucleotide polymer such as (AGA)n is used to program in vitro translation ---> the sequences of the resulting polypeptides are determined. •  The predicted codons are then correlated with the resulting proteins. •  Unambiguous assignments can only be made by repeating the experiment with a large number of nucleotide polymers. (See prob. 44 — note that the first line should be (UC)n not (UG)n •  H. Khorana finished breaking the code with this method and also won a Nobel prize. •  What’s the difference between this method and that used by Nirenberg and Matthaei? –  In the Nirenberg method, only one nucleotide was used. –  In the Khorana method, two and three different nucleotides were used to generate repeating sequences. Different polypeptides are produced because different reading frames are used. Downloaded by Soji Adimula ([email protected])

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The repeating nucleotide polymer approach Synthetic RNA: Clever chemistry allowed synthesis of short RNA oligonucleotides of known sequence

PNP + CU (CU)n, or CUCUCUCUCUCUCU … Two possible frames here: UCU-CUC-UCU-CUC or CUC-UCU-CUC-UCU In both cases, the protein produced has alternating serine and leucine: Ser-Leu-Ser-Leu-Ser-Leu … Khorana (problem 44) Downloaded by Soji Adimula ([email protected])

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Some synthetic RNAs failed to produce any protein: For example, (GAUA)n and (GUAA)n The reason – three codons do not encode any amino acids. These are UAA, UAG, and UGA, stop-codons GAUAGAUAGAUAGAUA GUAAGUAAGUAAGUAA Khorana (problem 44) Downloaded by Soji Adimula ([email protected])

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Cracking the Code #3: Identification of the Stop codons •  How Brenner deduced the role for UAG continued 1.  Brenner isolated 6 mutants of the T4 phage that had shortened head proteins. 2.  The mutant proteins were sequenced. 3.  By comparing the sequences of the mutant and wild type proteins, Brenner determined the identity of the amino acid that would have been inserted next in each of the mutants, had the mutations not occurred. 4.  In thinking about these six amino acids — glutamine, lysine, glutamic acid, tyrosine, and serine — Brenner deduced that each was specified by a codon that could mutate to UAG in a single step. Why is it important that UAG could be produced in a single step? 5.  For each of his six head mutants, Brenner isolated suppressor mutations in the host chromosome that restored wild type head length. How might these suppressors work? •  5'-UAC-3' codes for tyrosine, so it's anticodon is 3'-AUG-5'. A G --> C mutation will yield 3'-AUC-5' which recognizes the 5'-UAG-3' Stop codon. •  Question: Is this an intragenic or extragenic suppressor? Downloaded by Soji Adimula ([email protected])

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Cracking the Code #4: mini RNAs •  The code was fully deciphered using mini RNAs, and repeating nucleotide polymers. •  Mini RNAs are three nucleotides in length, of known sequence. •  The amino-acyltRNA that a given mini RNA binds in vitro is dictated by the code. –  Example: GUU binds valyl-tRNA in vitro. Therefore, GUU must code for valine. –  Note that this type of experiment alone could have been used to decipher the code. Why wasn’t it?

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How is the nucleic acid triplet code translated into a sequence of amino acids? Could the mRNA fold in such a way as to specify the amino acid sequence directly? •  Not likely. If mRNA is mixed with all 20 amino acids in a test tube, protein is not synthesized. What else is required? •  In 1958, Crick brilliantly hypothesized the existence of adapter molecules that fit the amino acids to the mRNA template, with the order of amino acids determined by nucleotide base pairing between the adapter and template. •  Crick speculated further that a distinct enzyme would be required to attach each adapter to its own amino acid.

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The adaptor is tRNA, which binds the amino acid to the codon and ribosome. Improper base pair?

• The 2˚ structure looks like a cloverleaf. • Note that the tRNA molecule has two "business ends." • Note that the anticodon is complementary and antiparallel to the codon.

Anticodon 5' to 3'

Modified nucleotides found only in tRNA: ψ, UH2, and mI (there are others) Downloaded by Soji Adimula ([email protected])

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The 3D structure of the various tRNAs is very similar, but not identical phenylalanine

glutamine

Unique shapes at the amino acyl end and the anticodon loop

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Aminoacyl-tRNA synthesis joins each amino acid with its tRNA(s)

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Aminoacyl-tRNA synthesis aax + tRNAx + ATP

aax-tRNAx + AMP + PPi

Ø  This reaction is catalyzed by the enzyme amnoacyltRNA synthetase. There are 20 such enzymes—1 for each amino acid. Ø  Each aminoacyl-tRNA synthetase recognizes a particular amino acid and attaches it to all tRNAs that carry that amino acid. Ø  Therefore, the accuracy (specificity) of protein synthesis depends on the ability of the enzyme to distinguish its particular amino acid and set of corresponding tRNAs from all other amino acids and tRNA species.

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Evidence that aminoacyl-tRNA synthesis provides the specificity of translation Experiment: chemically change the amino acid on a tRNA to another amino acid cysteine-tRNACys -------------> alanine-tRNACys Synthesize protein with alanine-tRNACys Result: Protein with alanines where the cysteines should be. Conclusion: The tRNA recognizes the codon, not the amino acid. The aminoacyl tRNA synthetases provides the specificity, not the ribosome. By joining the correct amino acids and tRNAs, the synthetases do the actual translation of the code!

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Degeneracy Revisited •  The genetic code is degenerate, which is another way of saying that it is redundant. This means that some amino acids are specified by more than one codon. How is this achieved? •  There are two distinct sources of degeneracy (redundancy) in the code: 1. Some aminoacyl tRNA synthetases couple their particular amino acid to more than one species of tRNA. In other words, tRNA molecules with different anticodons can carry the same amino acid. 2. In some cases, a given species of tRNA recognizes more than one codon, due to wobble.

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Wobble: An odd inexactitude

: In the 3rd position, G, U, and I can assume either of 2 positions, and thus can form unusual base pairs. Recall that H-bonds are highly directional.

•  Some tRNAs recognize more than one codon! Sloppy pairing (wobble) at the 3rd base of a codon allows one tRNA to recognize two or even three different codons. •  Notice that most codons for the same amino acid differ in the third nucleotide.

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Most codons for the same amino acid differ in the third nucleotide

Serine, leucince, and arginine are encoded by six codons.

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Rules of Wobble pairing Serine codons: UCU, UCC, UCA, UCG, AGU and AGC tRNASer1 3'-AGG-5' anticodon pairs with both UCU and UCC:

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Properties of the Genetic Code Summary The genetic code is nonoverlapping, uses three letter words that are read in a polarized fashion (in one direction), depends directly on tRNAs and the tRNA synthetases for specificity, and is highly degenerate (redundant).

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THE CODE

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•  Protein structure •  Genetic code •  Translation

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Protein synthesis: directed polymerization of amino acids •  What is a polymer? Polymers are large molecules composed of repeating structural units. •  What are some examples of polymers in biological systems? –  RNA & DNA –  Polypeptides (proteins) –  Glycogen –  Fats

•  Why is protein synthesis referred to as directed polymerization?

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Getting ready to polymerize These requirements arewe satisfied In order to polymerize, need: by . . . •  a mechanisnm for linking the subunits together the ribosome

•  a mechanism(s) that provides specificity the tRNA—aminoacyl tRNA synthetase interaction (and the anticodon)

•  energy to drive the reaction forward and thus allow a decrease in entropy ATP

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Charging the tRNA: a prelude to protein synthesis Charging the tRNA means to attach the aa to it. aax + tRNAx + ATP + aminoacyl-tRNA synthetasex

aax-tRNAx + AMP + PPi The energy provided by ATP is stored in the aa-tRNA bond

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Peptide bond formation (polymerization) aa1-tRNA1 + aa2-tRNA2

aa1- aa2-tRNA2 + tRNA1 Catalyzed by peptidyl transferase and the ribosome No additional energy required! Downloaded by Soji Adimula ([email protected])

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Dissection of the translational machinery •  The components of the translational machinery were identified by centrifugation through sucrose density gradients. •  This method separates molecules by the rate at which they move through the gradient, and thus according to their size and shape. –  The largest molecules reach the bottom of the tube first. These have the larger S value. (S stands for sedimentation coefficient.) –  Equilibrium is not reached.

•  In contrast, centrifugation through CsCl density gradients separates molecules according to their density (see chapter 7). Molecules band where they are as dense as the gradient. Equilibrium is reached. Downloaded by Soji Adimula ([email protected])

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Dissection of the translational machinery •  The translational machinery consists of several classes of macromolecule. –  tRNA (transfer RNA) –  mRNA (messenger RNA) –  Ribosomes •  Protein •  rRNA: 23S, 16S, 5S (ribosomal RNA)

•  You should know of these various components, but don't memorize details such as their exact sizes. •  tRNA and rRNA molecules are encoded by what?

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Ribosome Two subunits: large and small Both consist of RNA molecules and many protein molecules. About 2/3 of the ribosome’s mass is RNA.

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Ribosome rRNA molecules are huge and complex. The 16S prokaryotic rRNA from the small subunit of the ribosome is >1500 nucleotides long.

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Q: Why doesn't 50S + 30S = 80S and 60S + 40S = 100? Downloaded by Soji Adimula ([email protected])

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What you should know about the structure of ribosomes •  All ribosomes are composed of a large and a small subunit. –  The eukaryotic ribosomal subunits and assembled ribosome are slightly larger than their prokaryotic counterparts. –  The ribosomal subunits remain apart until the initiation of translation.

•  The ribosomal subunits are composed of both protein and RNA. –  Each subunit contains a large ribosomal RNA species. Thus, there are two large rRNAs in each ribosome. –  The large eukaryotic subunit contains two additional small rRNA species, whereas the large prokaryotic subunit only contains one. –  All ribosomal RNAs and proteins are encoded by genes. –  The ribosomal proteins are thought to be structural and the ribosomal RNAs are thought to be catalytic!

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What’s the goal here?

H

peptidyl

aminoacyl

Adapter

The ribosome reads the message in the 5' --> 3' direction. In what direction is the gene read? Downloaded by Soji Adimula ([email protected])

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Amino acids are connected by peptide bonds between the carboxyl end of one and the amino end of the next, thus forming a linear polypeptide chain.

Directionality!

Carboxyl end

Amino end Downloaded by Soji Adimula ([email protected])

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A site: entry of new Aminoacyl-tRNA P site: growing Peptide-tRNA (E Site: tRNA Exit)

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The three stages Just like transcription, translation can be divided into three stages: Ø  INITIATION Ø  ELONGATION Ø  TERMINATION Not to be confused with the three stooges: Larry, Moe, and Curly

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Initiation of translation — key points •  Two things are accomplished during initiation. –  The ribosome is assembled in a complex containing the mRNA and the first tRNA. –  The ribosome finds the initiation position (a particular AUG or GUG)

•  Initiation occurs in 3 steps, requiring 3 factors. –  The the small subunit of the ribosome binds to the mRNA. This step requires IF3 (initiation factor 3). –  N-formylmethionine binds to the P site of the ribosome. This step requires IF2-GTP. –  The large subunit binds to the complex, thus completing assembly. The energy for this reaction is derived from the hydrolysis of the GTP bound to IF2.

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Translation initiation in prokaryotes Initiation Factors IF1, IF2, and IF3 help the initiator tRNA position properly, then are replaced by the large subunit.

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Differences between initiation of translation in prokaryotes and eukaryotes •  Prokaryotes use fMet for initiation and methionine thereafter; eukaryotes use only methionine. •  Prokaryotes use AUG, GUG, and UUG as initiation codons; eukaryotes use only AUG. •  Prokaryotic messages are translated as they are being transcribed; eukaryotic messages must first be processed and transported to the cytoplasm. –  Prokaryotic messages are polycistronic (polygenic). Therefore, prokaryotic ribosome will have to find more than one initiation codon on each mRNA. –  Eukaryotic messages are monocistronic (monogenic), but have leader sequences and poly A tails.

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Polygenic vs. monogenic messages prokaryotic

eukaryotic

Moreover, there are multiple occurrences of AUG in any given message. How does the ribosome find the right AUG (and therefore the correct frame) to initiate translation? Prokaryotes and eukaryotes use different mechanisms.

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Specificity of initiation: How does the ribosome know where to start? •  John Shine and Lynn Dalgarno noticed that the true initiation codons are preceded by sequences that are complementary to the 3' end of the 16S rRNA. The interaction between this Shine and Dalgarno sequence and the 16S rRNA helps position the ribosome for proper initiation. Interaction between the S&D sequence and the 3' end of the 16S rRNA.

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Translation initiation in prokaryotes Initiation Factors IF1, IF2, and IF3 help the initiator tRNA position properly, then are replaced by the large subunit.

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Translation initiation in eukaryotes Initiation factors eIF4A, B, C bind to the 5'-Cap on the mRNA and recruit the small (40S) subunit. Together with initiator tRNA, this complex scans the mRNA sequence for the presence of AUG (START) codon.

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Translation initiation in eukaryotes continued.

Then eIF4A, eIF4B, and eIF4C are replaced by heavy (60S) subunit of the ribosome.

+ GTP

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Translation elongation •  Similar in prokaryotes and eukaryotes. •  Requires several Elongation Factors: EF-Tu, EF-Ts, and EF-G. •  In addition to the ATP used by the aminoacyl-tRNA synthetases, elongation requires about 3 more GTP molecules for each added amino acid.

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Translation elongation

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Translation elongation

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tRNA path: A→P→E Peptide path: P→A→P→A

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Translation termination •  The stop codons UAG, UGA, and UAA are not recognized by tRNAs, but by proteins called release factors. •  When the ribosome encounters a stop codon and the peptidyl-tRNA is in the P site (1), the release factors bind to the A site (2). This causes the polypeptide to be released and the ribosome to dissociate into its two subunits in a reaction driven GTP hydrolysis (3). •  The termination mechanism is similar in prokaryotes and eukaryotes. •  tRNAs with mutated anticodons can act as nonsense suppressors (see Fig. 9.18), but an abundance of such tRNAs can be detrimental to the cell. Why? Downloaded by Soji Adimula ([email protected])

1

2

3

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Protein Processing Many proteins are posttranslationally modified •  Phosphorylation – regulation of activity. Kinases add phosphates and phosphatases remove phosphates. •  Addition of lipids – helps proteins bind to the membrane •  Addition of short peptides (e.g., ubiquitin) – triggers degradation of proteins. •  Glycosylation – important for proteins on the cell surface (like blood group antigens) •  Splicing of proteins has been reported as well. •  Many other examples are known. Downloaded by Soji Adimula ([email protected])

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Protein targeting examples •  Secreted proteins or cell surface proteins are synthesized on the Endoplasmic Reticulum and sorted through the Golgi. •  Nuclear proteins have a special short amino-acid sequence called the NLS (Nuclear Localization Sequence) that triggers their transport to the nucleus. •  Most mitochondrial proteins are synthesized in mitochondria.

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Protein targeting Proteins that are needed synthesized on the Endoplasmic Reticulum

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Gene structure diagram Promoter: -35 -10

ATG +1 start (start codon)

Terminator stop codon

Translation

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Universality of the genetic code Information transfer is the same in all organisms: Replication DNA --> DNA Transcription DNA --> RNA Translation RNA --> protein RNA from any organism can be transcribed and translated in any other organism to give protein with the same sequence!

Conclusion: the genetic code and information transfer mechanisms are universal!

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End of chapter 9

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