translation brooker

Genetics: Analysis and PrinciplesRobert J. Brooker

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

CHAPTER 13

TRANSLATION OF mRNA

Protein synthesis

Proteins are the active participants in cell structure and function

Genes that encode polypeptides are termed structural genes These are transcribed into messenger RNA (mRNA)

The main function of the genetic material is to encode the production of cellular proteins In the correct cell, at the proper time, and in suitable

amountsCopyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13.1 THE GENETIC BASIS FOR PROTEIN SYNTHESIS

13-3

13-16Figure 13.2

Overview of gene expression

Note that the start codon sets the reading frame for all remaining codons


Translation involves an interpretation of one language into another In genetics, the nucleotide language of mRNA is

translated into the amino acid language of proteins

This relies on the genetic code Refer to Table 13.2

The genetic information is coded within mRNA in groups of three nucleotides known as codons

The Genetic Code

13-12

13-13Table 13-2


Special codons: AUG (which specifies methionine) = start codon

AUG specifies additional methionines within the coding sequence UAA, UAG and UGA = termination, or stop, codons

The code is degenerate More than one codon can specify the same amino acid

For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base

It is sometime referred to as the wobble base

The code is nearly universal Only a few rare exceptions have been noted

13-14


In the 1960s, Gobind Khorana and his collaborators developed a novel method to synthesize RNA They first created short RNAs (2 to 4 nucleotide long) that

had a defined sequence These were then linked together enzymatically to create

long copolymers Refer to Table 13.5

RNA Copolymers Helped to Crack the Genetic Code

13-26


Polypeptide synthesis has a directionality that parallels the 5’ to 3’ orientation of mRNA

During each cycle of elongation, a peptide bond is formed between the last amino acid in the polypeptide chain and the amino acid being added

The first amino acid has an exposed amino group Said to be N-terminal or amino terminal end

The last amino acid has an exposed carboxyl group Said to be C-terminal or carboxy terminal end

Refer to Figure 13.5

A Polypeptide Chain Has Directionality

13-29

13-30Figure 13.5

Carboxyl group Amino group

Condensation reaction releasing a

water molecule


13-31Figure 13.5

N terminal C terminal


13-32Figure 13.6


There are 20 amino acids that may be found in polypeptides Each contains a different side chain, or R group

Nonpolar amino acids are hydrophobic

They are often buried within the interior of a folded protein

13-33Figure 13.6


Nonpolar and charged amino acids are hydrophilic They are more likely to be on the surface of the protein


There are four levels of structures in proteins 1. Primary 2. Secondary 3. Tertiary 4. Quaternary

A protein’s primary structure is its amino acid sequence

Levels of Structures in Proteins

13-34

13-35Figure 13.7

The amino acid sequence of the

enzyme lysozyme

129 amino acids long

Within the cell, the protein will not be found in this linear state Rather, it will adapt

a compact 3-D structure

Indeed, this folding can begin during translation

The progression from the primary to the 3-D structure is dictated by the amino acid sequence within the polypeptide



The primary structure of a protein folds to form regular, repeating shapes known as secondary structures

There are two types of secondary structures helix sheet Certain amino acids are good candidates for each structure These are stabilized by the formation of hydrogen bonds

Refer to Figure 13.8


13-36


The short regions of secondary structure in a protein fold into a three-dimensional tertiary structure Refer to Figure 13.8 This is the final conformation of proteins that are

composed of a single polypeptide Structure determined by hydrophobic and ionic interactions as well as

hydrogen bonds and Van der Waals interactions

Proteins made up of two or more polypeptides have a quaternary structure: Also called Protein complex This is formed when the various polypeptides associate

together to make a functional protein Refer to Figure 13.8


13-37

13-38Figure 13.8


13-41

A comparison of phenotype and genotype at the molecular, organismal and cellular levels

Figure 13.9

Wild type mutant

In the 1950s, Francis Crick and Mahon Hoagland proposed the adaptor hypothesis tRNAs play a direct role in the recognition of codons in

the mRNA

In particular, the hypothesis proposed that tRNA has two functions 1. Recognizing a 3-base codon in mRNA 2. Carrying an amino acid that is specific for that codon


13.2 STRUCTURE AND FUNCTION OF tRNA

13-42

Recognition Between tRNA and mRNA


During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA

13-43Figure 13.10

tRNAs are named according to the

amino acid they bear

The anticodon is anti-parallel to

the codon


The secondary structure of tRNAs exhibits a cloverleaf pattern It contains

Three stem-loop structures; Variable region An acceptor stem and 3’ single strand region

The actual three-dimensional or tertiary structure involves additional folding

In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified nucleotides More than 60 of these can occur

tRNAs Share Common Structural Features

13-51

13-52Structure of tRNAFigure 13.12

Found in all tRNAs

Not found in all tRNAsOther variable sites are shown in blue as well

The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH2 = dihydrouridine m2G = dimethylguanosine = pseudouridine


The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases There are 20 types

One for each amino acid

Aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different molecules Amino acid, tRNA and ATP

Charging of tRNAs

13-53


The aminoacyl-tRNA synthetases are responsible for the “second genetic code”

The selection of the correct amino acid must be highly accurate or the polypeptides may be nonfunctional

Error rate is less than one in every 100,000 Sequences throughout the tRNA, including but not limited

to the anticodon, are used as recognition sites Many modified bases are used as markers

Charging of tRNAs

13-54

13-55Figure 13.13

The amino acid is attached to the 3’ end

by an ester bond

tRNA charging


As mentioned earlier, the genetic code is degenerate With the exception of serine, arginine and leucine, this

degeneracy always occurs at the codon’s third position

To explain this pattern of degeneracy, Francis Crick proposed in 1966 the Wobble hypothesis In the codon-anticodon recognition process, the first two

positions pair strictly according to the A – U /G – C rule However, the third position can actually “wobble” or move

a bit: ”wobble base / position” Thus tolerating certain types of mismatches

tRNAs and the Wobble Rule

13-56

13-57Wobble position and base pairing rulesFigure 13.14

tRNAs that can recognize the same codon are termed isoacceptor tRNAs

Recognized very poorly by the tRNA

5-methyl-2-thiouridine

inosine

5-methyl-2’-O-methyluridine

5-methyluridine

lysidine

2’-O-methyluridine

5-hydroxyuridine


Translation occurs on the surface of a large macromolecular complex termed the ribosome

Bacterial cells have one type of ribosome Found in their cytoplasm

Eukaryotic cells have two types of ribosomes One type is found in the cytoplasm The other is found in organelles

Mitochondria ; Chloroplasts


13.3 RIBOSOME STRUCTURE AND ASSEMBLY

13-58

Unless otherwise noted the term eukaryotic ribosome refers to the ribosomes in the cytosol

A ribosome is composed of structures called the large and small subunits Each subunit is formed from the assembly of

Proteins rRNA

Figure 13.15 presents the composition of bacterial and eukaryotic ribosomes


13.3 RIBOSOME STRUCTURE AND ASSEMBLY

13-59

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-60Figure 13.15

Note: S or Svedberg units are not additive

Synthesis and assembly of all ribosome components occurs in the cytoplasm

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-61Figure 13.15

The 40S and 60S subunits are assembled in the nucleolus

Then exported to the cytoplasm

Synthesized in the

nucleus

Produced in the cytosol

Formed in the cytoplasm during

translation


During bacterial translation, the mRNA lies on the surface of the 30S subunit As a polypeptide is being synthesized, it exits through a

hole within the 50S subunit

Ribosomes contain three discrete sites Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site)

Ribosomal structure is shown in Figure 13.16

Functional Sites of Ribosomes

13-62

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-63

Figure 13.16

Translation can be viewed as occurring in three stages Initiation Elongation Termination

Refer to 13.17 for an overview of translation

13.4 STAGES OF TRANSLATION

13-64

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFigure 13.17

Initiator tRNA

See animation on your CD


The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex This process requires three Initiation Factors

The initiator tRNA recognizes the start codon in mRNA In bacteria, this tRNA is designated tRNAfmet

It carries a methionine that has been covalently modified to N-formylmethionine

The start codon is AUG, but in some cases GUG or UUG In all three cases, the first amino acid is N-formylmethionine

The Translation Initiation Stage


The binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site or Shine-Dalgarno sequence

This is complementary to a sequence in the 16S rRNA

13-67

Figure 13.18 outlines the steps that occur during translational initiation in bacteria

Figure 13.19

Hydrogen bonding

Component of the 30S subunit

13-68Figure 13.18


13-69Figure 13.18

70S initiation complex

This marks the end of the first

stage

The only charged tRNA that enters

through the P site

All others enter through the A site



In eukaryotes, the assembly of the initiation complex is similar to that in bacteria However, additional factors are required

Note that eukaryotic Initiation Factors are denoted eIF

The initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine

The Translation Initiation Stage

13-70


The start codon for eukaryotic translation is AUG It is usually the first AUG after the 5’ Cap

The consensus sequence for optimal start codon recognition is show here

13-71

Start codon

G C C (A/G) C C A U G G

-6 -5 -4 -3 -2 -1 +1 +2 +3 +4

Most important positions for codon selection

These rules are called Kozak’s rules After Marilyn Kozak who first proposed them

With that in mind, the start codon for eukaryotic translation is usually the first AUG after the 5’ Cap!

Translational initiation in eukaryotes can be summarized as such:

A number of initiation factors bind to the 5’ cap in mRNA These are joined by a complex consisting of the 40S

subunit, tRNAmet, and other initiation factors The entire assembly moves along the mRNA scanning

for the right start codon

Once it finds this AUG, the 40S subunit binds to it The 60S subunit joins This forms the 80S initiation complex

After the protein is synthesised the first aminoacid is removed! Final protein is 1 aa shorther than the number of aa codons in a mRNA! 13-72


During this stage, the amino acids are added to the polypeptide chain, one at a time

The addition of each amino acid occurs via a series of steps outlined in Figure 13.20

This process, though complex, can occur at a remarkable rate In bacteria 15-18 amino acids per second In eukaryotes 6 amino acids per second

The Translation Elongation Stage

13-73

13-74Figure 13.20

The 23S rRNA (a component of the large subunit) is the actual

peptidyl transferase

Thus, the ribosome is a ribozyme!


13-75Figure 13.20

tRNAs at the P and A sites move into the

E and P sites, respectively


Max. 2 tRNAs bound to mRNA in a ribosome !

Peptide bond formation is catalyzed by rRNA, not by one of the proteins in the ribosome!


16S rRNA (a part of the 30S ribosomal subunit) plays a key role in codon-anticodon recognition It can detect an incorrect tRNA bound at the A site

It will prevent elongation until the mispaired tRNA is released

This phenomenon is termed the decoding function of the ribosome It is important in maintaining the high fidelity in mRNA

translation Error rate: 1 mistake per 10,000 amino acids added

The Translation Elongation Stage

13-76


The final stage occurs when a stop codon is reached in the mRNA In most species there are three stop or nonsense codons

UAG UAA UGA

These codons are not recognized by tRNAs, but by proteins called release factors

Indeed, the 3-D structure of release factors mimics that of tRNAs

The Translation Termination Stage

13-77


Bacteria have three release factors

RF1, which recognizes UAA and UAG RF2, which recognizes UAA and UGA RF3, which does not recognize any of the three codons

It binds GTP and helps facilitate the termination process

Eukaryotes only have one release factor

eRF, which recognizes all three stop codons

The Translation Termination Stage

13-78

13-79Figure 13.21


Bacteria lack a nucleus Therefore, both transcription and translation occur in the cytoplasm

As soon an mRNA strand is long enough, a ribosome will attach to its 5’ end

So translation begins before transcription ends This phenomenon is termed coupling

A polyribosome or polysome is an mRNA transcript that has many bound ribosomes in the act of translation

Bacterial Translation Can Begin Before Transcription Is Completed

13-81

Messenger RNA Reading Frame- Each mRNA can be read in 3 ‘frames’

- Sequence of codons is important: start codon indicates in which ‘frames’ should be read

Sequence of Triplet form the genetic code and the mutations in a

gene :

Frameshift Mutations:By insertion/deletion of 1 or 2

nucleotiden

Restoration of Frame shift

Mutations:Base changes

Very harmful :

- deletion / insertion of one base- deletion / insertion of two basesEspecially in the beginning of a ORF/geneIf at the end of a gene, the ‘shorter’ (or the to the-C-terminal-end-aberrant) protein can still be active

Less harmful :- base substitution (another amino acid; but can also become a stop codon)- deletion / insertion of three bases (loss of an extra amino acid)

End of Chapter 13

translation brooker

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