Gene Expression at the

Molecular Level I:
Production of mRNA and Proteins
Chapter
12
 Chapter 12 Outline
• Overview of Gene Expression

• Transcription

• RNA Modification in Eukaryotes

• Translation and the Genetic code

• The Machinery of Translation

• The Stages of Translation

2
 Gene Expression & Mutation
 Gene expression is the process by which the information of a gene is made into a
functional product
 Studied at both the molecular level & the trait level

 A mutation is a heritable change in the genetic material

 Can affect gene function by altering the gene sequence

 Research focused on the effects of mutations proved instrumental in demonstrating

the relationships between:
 Normal genes and functional proteins
 Mutated abnormal genes and non-functional proteins
12.1 Overview of Gene Expression 3
 Inborn Errors of Metabolism
 Archibald Garrod studied patients with metabolic defects such as alkaptonuria
 Inherited disease in which patient’s body accumulates abnormal levels of
homogentisic acid (alkapton)

 In 1908, Garrod proposed a relationship between inheritance of a mutant gene, a

missing or defective enzyme, and a metabolic disease

 Called this an inborn error of metabolism

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Phenylalanine
metabolism

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 Beadle and Tatum
 Discovered Garrod’s work in the early 1940s

 Studied Neurospora crassa, common bread mold

 Minimum requirements for growth are carbon source (sugar), inorganic salts, and
biotin
 Many different cellular enzymes are used to synthesize all molecules needed for
growth from these components

 Focused on amino acid synthesis

 Hypothesized that genes encode enzymes, and a mutation might cause a defect in an
enzyme needed for amino acid synthesis
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 Beadle and Tatum
 Proposed that a different enzyme catalyzes each step in a biochemical pathway
and that a different gene controls the production of each different enzyme

 Collected different mutant strains that required supplementation with the amino
acid arginine for growth
 The pathway for arginine synthesis was thought to involve at least three precursor
molecules
 Therefore, three genes & three enzymes should be involved

 Examined the ability of mutant strains to grow in the presence of the different
precursor molecules
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 Beadle and Tatum
 Mutant strains fell into three groups according to which enzyme was defective
 Concluded that a single gene controls the synthesis of a single enzyme
 The one gene – one enzyme hypothesis

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 Not Quite…
 Enzymes are only one category of cellular proteins
 Genes also encode for many other types of proteins

 Some proteins are composed of two or more polypeptides

 E.g. Hemoglobin is composed of two α-globin and two β-globin polypeptides

 Some mRNAs can be spliced in alternative ways

 Allows a single gene to encode more than one polypeptide

 Some genes encode RNAs that are not used to make polypeptides
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The Central Dogma of
Gene Expression

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Prokaryotes vs. Eukaryotes

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 Genes
 A gene is an organized unit of DNA sequences that is transcribed into RNA and
results in the formation of a functional product

 Protein-coding genes are transcribed to produce a messenger RNA (mRNA)

that specifies the amino acid sequence of a polypeptide
 The polypeptide is considered to be the functional product
 The mRNA is an intermediary in polypeptide synthesis

 For non-coding RNA genes, the RNA itself is the final functional product
 e.g. Transfer RNA (tRNA) and ribosomal RNA (rRNA)

12.2 Transcription 12
Gene Organization

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Three Stages of Transcription

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 A Closer Look at Elongation
 RNA is synthesized in the 5’ to 3’ direction
 Complementarity between ribonucleotides and DNA template guides sequence of RNA
 Uracil substitutes for thymine

 The strand of DNA that is used as a template for RNA synthesis is called the template or
noncoding strand
 The opposite DNA strand that is not used for transcription is called the coding strand
 Has the same sequence as the mRNA (except T instead of U)

 Unlike DNA polymerase:

 RNA polymerases can start on a template without a primer
 RNA polymerases do not proofread and have no exonuclease activity
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Coding
strand

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 Transcription along the chromosome
 The DNA strand used as a template can vary for adjacent genes
 Depends on position of promoter sequences
 RNA is always synthesized 5’ to 3’
 Template strand is read in the 3’ to 5’ direction

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 Eukaryotic Transcription
 Basic features of transcription are
identical between prokaryotic and
eukaryotic organisms, but each step
tends to involve a greater complexity
of protein components

 E.g. Eukaryotes have three RNA

polymerases instead of one, and
initiation of transcription requires
five general transcription factors
instead of sigma factor

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 Eukaryotic RNA Processing

12.3 RNA Modification in Eukaryotes 19

 RNA Processing: Capping
 Covalent attachment of 7-methylguanosine
to the 5’ end of the mRNA transcript
 = 5’ cap

 Occurs while RNA polymerase is still

creating the pre-mRNA

 5 cap is recognized by cap-binding proteins

 Required for proper exit of the mRNA from
the nucleus
 Protects mRNA and helps it bind to a
ribosome for translation

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 RNA Processing: Tailing
 Poly adenylation sequence in mRNA
attracts enzyme complex that cuts
mRNA and adds 100 to 200 adenines
to the 3’ end
 = poly A tail

 Aids in export from nucleus and

increases mRNA stability
 Allows mRNA to exist for a longer time
in the cytosol

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 RNA Processing: Splicing
 Many eukaryotic genes contain coding sequences that are interrupted by large
segments of DNA that are transcribed but not translated
 Exons are the RNA sequences found in the mature mRNA
 Introns are intervening untranslated sequences

 Splicing removes introns and joins exons together

 Catalyzed by the spliceosome
 Consists of several subunits (snRNPs) made of snRNA & protein

 In alternative splicing, splicing can occur more than one way to produce different
products
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23
 The Genetic Code
 Specifies the relationship between the sequence of nucleotides in mRNA and the
sequence of amino acids in a polypeptide

 Sequence of an mRNA is read in groups of three consecutive ribonucleotide

bases = codons
 Specify for a particular amino acid, or indicate start or stop
 e.g. CCC = proline, GGC = glycine
 tRNAs bind to mRNA codons using anticodons

12.4 Translation and the Genetic Code 24

 The Genetic Code
 Using 3 bases, 64 different codons are
possible (= 43)
 Only 20 different amino acids

 Genetic code is degenerate

 More than one codon can specify the
same amino acid

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Bacterial mRNA Organization

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 Reading Frame
 The start codon defines the reading frame of an mRNA
 Each adjacent codon is read as a triplet in the 5 to 3 direction

 Insertion or deletion of any number of bases that is not a multiple of 3 changes the amino acid
sequence

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28
 Feature Investigation

Nirenberg & Leder

• In 1964, Nirenberg and Leder discovered that an RNA triplet can act like a codon
within an mRNA molecule

• Made different RNA molecules with specific triplet sequences

• E.g. 5’-CCC-3’ triplets

• Each RNA underwent in vitro translation in 20 different tubes

• Each tube had a different radiolabeled amino acid
• By detecting which radioactive amino acid was linked to the triplet, could determine what
amino acid is encoded by each triplet sequence
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30
31
 The Translation Machinery

The Translation
Machinery

12.5 The Machinery of Translation 32

 tRNA
 Different tRNAs are encoded by
different genes

 Common features
 Cloverleaf structure
 3’ single-stranded region for amino
acid binding = Acceptor stem
 Anticodon

 Notation indicates amino acid

 tRNAser carries serine
 Aminoacyl-tRNA Synthetases
 Enzymes that catalyze the attachment of amino acids to tRNA molecules (=
Charging a tRNA)
 Cells contain 20 different aminoacyl-tRNA synthetases
 One for each of the 20 different amino acids

 Two-step reaction results in a tRNA with an amino acid attached

 = Charged tRNA or aminoacyl tRNA

 Highly accurate process

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35
 Ribosomes
 Macromolecular site where translation takes place

 Composed of many different proteins & RNAs assembled into large and small
subunits
 Eukaryotic ribosomes consist of 40S and 60S subunits that combine to form an 80S
ribosome
 Prokaryotes have 30S and 50S subunits that form a 70S ribosome

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 RNA
Ribosome Structure protein

 Overall ribosome shape determined

by rRNAs

 tRNAs bind to the ribosome at three

discrete sites:
 A site = aminoacyl site
 P site = peptidyl site
 E site = exit site
 rRNAs & Evolutionary Relationships
 Comparisons of small subunit rRNAs among different species provide a basis for
establishing evolutionary relationships
 The gene for the small subunit rRNA is found in the genomes of all organisms
 At the molecular level, gene evolution involves changes in DNA sequences
 If a long time has elapsed since two species diverged evolutionarily, their genes tend
to be more different than closely related species

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Evolutionary Relationships

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 Three Stages of Translation

12.6 The Stages of Translation 40

Translation Initiation in
Bacteria

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 Eukaryotic Initiation
Two key differences:
 mRNAs do not contain a ribosomal binding site
 Cap-binding proteins bind to 5’ cap, and promote binding of mRNA to small
ribosomal subunit

 Position of start codon is more variable

 Small ribosomal subunit begins at 5’ cap and scans the mRNA
 In many cases, first AUG codon is used as the start codon

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 Elongation – 1
1. Aminoacyl tRNA carrying a single amino
acid binds to the A site
 Via codon / anticodon recognition
 Peptidyl tRNA (attached to polypeptide)
is in the P site
 Aminoacyl tRNA is in the A site

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 Elongation – 2
2. A peptide bond is formed between
the amino acid at the A site and the
polypeptide chain
 Catalyzed by rRNA E P A

 Ribosome is a ribozyme
 Polypeptide transfers from tRNA in P
site to amino acid in A site
 = Peptidyl transfer reaction

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 Elongation – 3
3. Ribosome moves (translocates) toward
the 3’ end of the mRNA by one codon
 Shifts tRNAs down by one site
 Uncharged tRNA transfers from P site E P A
into E site, where it exits the ribosome
 tRNA containing the polypeptide moves
from A site into P site
 The next codon is now exposed at an
unoccupied A site

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 Termination
 Translation ends when a stop codon is
found in the A site
 Recognized by proteins known as
release factors

 Binding of release factor to stop

codon causes bond between
polypeptide and tRNA in P site to
break
 Releases polypeptide and tRNA
 Remaining subunits dissociate

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 Antibiotics
 Many antibiotics that treat
bacterial infections inhibit or
interfere with bacterial
translation, without affecting
eukaryotic translation

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