Transcript PowerPoint Presentation Materials to accompany

PowerPoint Presentation Materials
to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 13 Part 2
TRANSLATION OF mRNA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13.2 STRUCTURE AND
FUNCTION OF tRNA

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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-38
Recognition Between tRNA and mRNA

During mRNA-tRNA recognition, the anticodon in
tRNA binds to a complementary codon in mRNA
tRNAs are named
according to the
amino acid they bear
Proline
anticodon
Figure 13.8
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-39
Experiment 13B: The Adaptor
Hypothesis Put to the Test


1962, François Chapeville
Hypothesis: the amino acid attached to tRNA is not
directly involved in codon recognition


Therefore, the alteration of an amino acid already attached
to tRNA should cause that altered amino acid to be
incorporated into the polypeptide instead of the normal
amino acid
Example: Cysteine on a tRNAcys is changed to alanine
cys will add alanine instead of the
 Therefore, the tRNA
usual cysteine
13-40
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Raney nickel converts cysteine to alanine

The experiment made use of a cell-free translation system
similar to the one used by Nirenberg

Refer to Figure 13.3

Chapeville used an mRNA template that contained only U and G


Therefore, it could only contain the following eight codons
UUU = phenylalanine
GUU = valine
UUG = leucine
GUG = valine
UGU = cysteine
GGU = glycine
UGG =tryptophan
GGG = glycine
Note: One cysteine codon and no alanine codons
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-41
The Hypothesis

Codon recognition is dictated only by the tRNA

The chemical structure of the amino acid attached to
tRNA does not play a role
Testing the Hypothesis

Refer to Figure 13.9
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-42
Figure 13.9
13-43
Figure 13.9
13-44
The Data
Relative Amount of Radiolabeled
Amino Acid Incorporated into
Polypeptide (cpm)*
Conditions
Control, untreated tRNA
Raney nickel-treated tRNA
Cysteine
Alanine
Total
2,835
83
2,918
990
2,020
3,010
*Cpm is counts per minute of radioactivity in the sample
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-45
Interpreting the Data
Relative Amount of Radiolabeled
Amino Acid Incorporated into
Polypeptide (cpm)*
Conditions
Control, untreated tRNA
Raney nickel-treated tRNA
Cysteine
Alanine
Total
2,835
83
2,918
990
2,020
3,010
Expected result
since only
radiolabeled
cysteine was added
Probably a result
of cysteine
contamination
*Cpm is counts per minute of radioactivity in the sample
About a third of the
tRNAcys did not react
with the Raney nickel
Large amount of incorporated
alanine even though template
mRNA lacks alanine codons
Overall, these results support the adaptor hypothesis
tRNAs act as adaptors to carry the correct amino acid to
the ribosome based on their anticodon sequence
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-46
tRNAs Share Common Structural
Features

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
In addition to the normal A, U, G and C nucleotides,
tRNAs commonly contain modified nucleotides

More than 60 of these can occur
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-47
Found in all tRNAs
Not found in all tRNAs
Other variable sites are
shown in blue as well







Figure 13.10 Structure of tRNA
The modified bases are:
I = inosine
mI = methylinosine
T = ribothymidine
UH2 = dihydrouridine
m2G = dimethylguanosine
y = pseudouridine
13-48
Transfer RNA
• Molecules of tRNA are not all identical
– Each carries a specific amino acid on one end
– Each has an anticodon on the other end
– Fidelity of translation of the genetic code is
determined at two levels
• Linkage of specific a.a. to specific tRNA
• mRNA codon:tRNA anti-codon recognition
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5
3
Amino acid
attachment site
Hydrogen
bonds
A
3
Anticodon
(b) Three-dimensional structure
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
A G
Anticodon
5
(c) Symbol used
in this book
Charging of tRNAs

The enzymes that attach amino acids to tRNAs are
known as aminoacyl-tRNA synthetases

There are 20 types

One for each amino acid
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-49
The amino acid is
attached to the 3’ end
by an ester bond
Figure 13.11
13-50
tRNAs and the Wobble Rule

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

Thus tolerating certain types of mismatches
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-51
tRNAs that can recognize the same
codon are termed isoacceptor tRNAs

inosine
5-methyl-2-thiouridine
5-methyl-2’-O-methyluridine

2’-O-methyluridine

5-methyluridine

5-hydroxyuridine

lysidine


Recognized very
poorly by the tRNA
Figure 13.12 Wobble position and base pairing rules
13-52
tRNAs and the Wobble Rule




Wobble allows for economizing:
61 codons specifying amino acids
Bacteria: 30 to 40 tRNAs
Eukaryotes: up to 50 tRNAs
13.3 RIBOSOME STRUCTURE
AND ASSEMBLY

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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-53
13.3 RIBOSOME STRUCTURE
AND ASSEMBLY

A ribosome is composed of structures
called the large and small subunits

Each subunit is formed from the assembly of



Proteins
rRNA
Figure 3.13 presents the composition of
bacterial and eukaryotic ribosomes
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-54
Synthesis and assembly of all ribosome
components occurs in the cytoplasm
(a) Bacterial cell
Note: S or Svedberg units
are not additive
Figure 13.13
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-55
Synthesized in
the nucleus
Formed in the
cytoplasm during
translation
Produced in the
cytosol
The 40S and 60S subunits are
assembled in the nucleolus
Then exported to the cytoplasm
Figure 13.13
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-56
13.4 STAGES OF
TRANSLATION

Translation can be viewed as occurring in three
stages




Initiation
Elongation
Termination
Refer to 13.15 for an overview of translation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-59
Initiator tRNA
Release
factors
Figure 13.15
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-60
The Translation Initiation Stage

The mRNA, initiator tRNA, and ribosomal subunits
associate to form an initiation complex

The initiator tRNA recognizes the start codon AUG in
mRNA

In bacteria, this tRNA is designated tRNAfmet


It carries a methionine that has been covalently modified to
N-formylmethionine
In eukaryotes, the initiator tRNA is designated tRNAmet

It carries a methionine rather than a formylmethionine
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-61

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
Hydrogen bonding
Component of the
30S subunit
Figure 13.17

16S rRNA
Figure 13.16 outlines the steps that occur during
translational initiation in bacteria
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-62
(actually 9
nucleotides long)
Figure 13.16
13-63
The only charged
tRNA that enters
through the P site
All others enter
through the A site
70S initiation
complex
This marks the
end of the first
stage
Figure 13.16
13-64

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
Most important positions for codon selection


C C A U G G
-2 -1 +1 +2 +3 +4
These rules are called Kozak’s rules


G C C (A/G)
-6 -5 -4
-3
Start codon
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!
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-66

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 complex 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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-67
The Translation Termination Stage

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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-72
The ribosomal subunits and
mRNA dissociate
Figure 13.19
13-74
A Polypeptide Chain Has
Directionality

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

Refer to Figure 13.20
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-76
Carboxyl group
Amino group
Condensation
reaction releasing a
water molecule
Figure 13.20
13-77
N terminal
Figure 13.20
C terminal
13-78