Chapter 12

Gene Regulation at the Molecular Level

OR
Transcription and Translation!
How does the cell read the
information contained in the
Genomic DNA?
 DNA is a linear polymer made of
nucleotides
 The order of the nucleotides carries the
information needed to make gene products
(anything that a gene encodes - tRNA,
rRNA, mRNA --> proteins,etc.)
What makes up a Gene?
 It’s a region/sequence of DNA

 Always have a promoter

 Always have transcribed region
• For genes encoding proteins the transcript will
contain a contain a “coding region”
 Always have a terminator
Gene expression
 level of traits (cellular or organismic level)
 molecular level

 Interconnection between two levels

• molecular level determines the structure and
function of cells  traits expressed

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How do genes work?
 Mendel came along with the idea of a
particle carrying genetic information
 Garrod postulated that genes coded for
enzymes
• Patients with metabolic defects
• Disease due to missing enzyme
• Recessive inheritance
• Inborn error of metabolism
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 Beadle and Tatum studied Neurospora a
fungi
• Strains defective in Amino Acid Synthesis
• Arginine auxotrophs - can’t make arginine
 Isolated mutants
 Examined for ability to grow in the presence of precursors
 3 classes of mutants depending on nutrient required
• One gene – one enzyme hypothesis
• Not quite right, but on right track!

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 One gene - one polypeptide
• Not all genes are enzymes….
• Hemoglobin composed of 4 polypeptides
required for function
• 2  polypeptides
• 2  polypeptides
 Not quite right either!
• Alternate splicing
• More on this later
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 Central dogma
 DNA RNA  Protein
 Transcription
• Synthesis of RNA from a DNA template
 Translation
• Synthesis of polypeptides from an RNA transcript on a
ribosome

• Proteins (DNA polymerase etc.) can then be used to replicate DNA

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 Genes – information to make organism
 Structural genes code for polypeptides
• Polypeptide folds into a protein
• OR folds with other polypeptides to make a
protein
 Proteins determine structure and function of
cells
 Traits or characteristics of organism based on
cellular activities due to proteins

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Transcription
 Copying DNA into an RNA molecule of some
sort
 Over 90% of all genes are structural (mRNA
proteins)
 Other genes code for the RNA itself
• Transfer RNA (tRNA) – helps in translation of
mRNA into amino acids
• Ribosomal RNA (rRNA) - part of ribosomes
• Other types of RNA too…..scRNA, snoRNA etc.

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Three stages
1. Initiation
2. Elongation
3. Termination

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Three stages
1. Initiation
• Recognition of start site
• In bacteria, sigma factor causes RNA polymerase
to recognize promoter region
• w/o sigma factor, RNA polymerase is “promiscuous”
• Catalytic portion of RNA polymerase has similar
structure in all species
• NOTE RNA polymerases do NOT need a primer

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2. Elongation
• RNA polymerase synthesizes RNA after release of sigma
factor
• Open complex 10-15 base pairs long
• Template or non-coding strand used for RNA synthesis
• Coding (sense) strand is not used
• Synthesized 5’ to 3’
• Uracil substituted for thymine
• Behind open complex, DNA rewinds

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3. Termination
• RNA polymerase reaches termination
sequence
• RNA polymerase falls off DNA
• New (nascent) RNA molecule is released

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 Neighboring genes may have opposite orientations
• Different strand of DNA is used as template
 RNA synthesis is 5’ to 3’
• Just as we saw for DNA synthesis

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Eukaryotic transcription
 Nearly identical to prokaryotes
 But more proteins involved
 3 forms of RNA polymerase
• RNA polymerase II – transcribes mRNA
• RNA polymerase I and III – transcribes
nonstructural genes for rRNA and tRNA
 RNA polymerase II
• 5 initiation factors
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 RNA processing
 Bacterial mRNAs
• translated into polypeptides immediately

 Eukaryotic mRNAs
• pre-mRNA  mature mRNA
• Introns- transcribed but not translated  removed before export to cytosol
• Exons- coding sequence found in mature mRNA
 Processing
• Splicing- removal of introns and connection of exons
• 7-methyl G cap addition
• PolyA tail

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Splicing
 Leder’s experiment to identify introns in the
β-globin gene
 Double stranded DNA of the β-globin gene
denatured + mature mRNA of the β-globin
gene
 If mRNA binds to a region of a gene with
one intron  two single stranded DNA loops
separated by a double-stranded DNA region

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 Introns found in many eukaryotic genes
 Widespread in complex eukaryotes
• Human dystrophin gene has 79 exons and 78 introns
 Very Few introns in prokaryotes
 Introns removed from eukaryotic pre-mRNA using a
spliceosome composed of snRNPs (snurps) – Joan
Stietz
• Nuclear RNA and a set of proteins

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 Intron RNA is defined by particular sequences within
the intron and at the intron-exon boundaries
• 5’ splice site, branch site, 3’ splice site
 Spliceosome subunits recognize intron sequences
 Binding causes intron to loop outward
• Lariat structure released
 Two exons brought closer together and joined
• Alternative splicing- spliceosome regulated so that single
gene can encode 2 or more polypeptides
 Introns in rRNA and tRNA are self-splicing

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 5’ cap and capping
• 7-methylguanosine (7-methyl G cap) covalently
attached to 5’ end
• Export from nucleus
• Initiation of translation
 3’ poly A tail
• 100-200+ adenine nucleotides added to 3’ end
• Increases stability and lifespan in cytosol
• Not encoded in gene sequence
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 Translation
 Genetic code – sequence of bases in an mRNA
molecule  amino acid sequence
 Read in groups of three nucleotide bases or
codons
• 64 codons
• 20 amino acids  some amino acids encode by >1
codon
• 1 start, 3 stop codons

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 Bacterial mRNA
• 5’ ribosomal-binding site
Shine-Delgarno
• Start codon usually AUG
• Need to know!
• Tells where to start the
polypeptide
• Reading Frame!!!!!

 1 of 3 stop codons
• UAA, UAG or UGA
• Don’t need to know these
exact codons

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Reading frame
 Start codon defines reading frame
5’ –AUAAGGAGGUUACG(AUG)(CAG)(CAG)(GGC)(UUU)(ACC) – 3’
Met –Gln -Gln -Gly -Phe -Thr

 Addition of a U shifts the reading frame and

changes the codons and amino acids
specified
5’ –AUAAGGAGGUUACG(AUG)(UCA)(GCA)(GGG)(CUU)(UAC)C – 3’
Met –Ser -Ala -Gly -Leu -Tyr

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 tRNA – encoded by tRNA genes
• Anticodon allows binding of tRNA to mRNA
codon
• Hydrogen bonds
• Many modified nucleotides – not just GAUC

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DNA, RNA and Protein are co-linear

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Nirenberg and Leder found the RNA triplets can
promote the binding of tRNA to ribosomes
 1964 found that an RNA triplet can act like a
codon within an mRNA molecule
 Experiment establishes relationship between
triplet sequence and specific amino acids
 Used radiolabeled amino acids bound to tRNA
 Complex of tRNA, RNA triplet and ribosome
could be filtered by size
 tRNA
 Different tRNA
molecules encoded by
different genes
 tRNAser carries serine
 Common features
• Cloverleaf structure
• Anticodon
• Acceptor stem for
amino acid binding

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 Aminoacyl-tRNA synthetases catalyze the
attachment of amino acids to tRNA
 2 reactions result in tRNA with amino acid
attached (charged tRNA, aminoacyl tRNA)
 Ability of aminoacyl-tRNA synthetase to
recognize appropriate tRNA has been
called the second genetic code
 Fidelity very important!!!

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Charging of a tRNA!

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 Ribosomes
 Prokaryotes - only one type
 Eukaryotes – 2-3 types (cytosolic, mitochodrial,
chloroplast)
• Focus on cytosolic ribosomes
 Composed of a large and a small subunit
• Proteins + rRNAs
 Structural differences between prokaryotes and eukaryotes
• antibiotics to inhibit bacterial ribosomes only

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 Overall ribosome
shape determined by
rRNA – bases highly
modified
 P site- peptidyl site
 A site- aminoacyl
site
 E site- exit site

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 Comparisons of small subunit rRNAs among
different species provide basis for establishing
evolutionary relationships
 All organisms have evolutionarily related
translational components
 Gene for small subunit rRNA (SSU rRNA) found in
all genomes
 Gene evolution involves changes in DNA sequences
 Identical sequences are evolutionarily conserved
• Critical function not subject to change
 Gene sequences more similar in more closely
related species
Stages of Translation
1. Initiation
• mRNA, first tRNA and ribosomal subunits
assemble
2. Elongation
• Synthesis from start codon to stop codon
3. Termination
• Complex disassembles at stop codon
releasing completed polypeptide

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Initiation
 mRNA, first tRNA and ribosomal subunits
assemble
 Requires help of ribosomal initiation factors
 Also requires input of energy (GTP
hydrolysis)

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 Bacteria
• mRNA binds to small ribosomal subunit
facilitated by ribosomal-binding sequence
• Start codon a few nucleotides downstream
• Initiator tRNA recognizes start codon in mRNA
• Large ribosomal subunit associates
• At the end, the initiator tRNA is in the P site

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 Eukaryotic differences in initiation
• Instead of a ribosomal-binding sequence,
mRNAs have 7-methylguanosine cap at 5’ end
• Recognized by cap-binding proteins
• Small subunit binds to cap + initiation factors
– Scans down to start codon – requires energy
• Position of start codon more variable
• In many cases, first AUG codon used as start codon
• Not always – nearby sequences are very important

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Elongation
1. Aminoacyl tRNA brings a new amino acid
to the A site
• Binding occurs due to codon/ anticodon
recognition
• Elongation factors hydrolzye GTP to provide
energy to bind tRNA to A site
• Peptidyl tRNA is in the P site
• Aminoacyl tRNA is in the A site

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2. A peptide bond is formed between the
amino acid at the A site and the growing
polypeptide chain
 The polypeptide is removed from the tRNA in
the P site and transferred to the amino acid at
the A site – peptidyl transfer reaction

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3. Movement or translocation of the
ribosome toward the 3’ end of the mRNA
by one codon
• Shifts tRNAs at the P and A sites to the E and
P sites
• The next codon is now at the A spot
• Uncharged tRNA exits from E spot

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Termination
 When a stop codon is found in the A site,
translation ends
 3 stop codons- UAA, UAG, UGA
 Recognized by release factors
 Can’t make a peptide bond

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 Completed polypeptide attached to a tRNA in
the P site and stop codon in the A site
1. Release factor binds to stop codon at the A site
2. Bond between polypeptide and tRNA
hydrolyzed to release polypeptide
3. Ribosomal subunits and release factors
disassociate

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 Polypeptide chain has directionality
 Parallels 5’ to 3’ orientation of mRNA
 N-terminus or amino terminus is first amino acid
 C-terminus or carboxyl terminus is last amino acid

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Transcription and Translation at the
same time!
 Bacteria don’t
have a nuclear
membrane

 Transcription
and
Translation
can be
coupled