Basic Biology
Chapter 12
Gene Expression at the Molecular Level
Prof. Ranjit Vijayan
Department of Biology
ranjit.v@[Link]
03 713 6302
Central Dogma: From DNA to RNA to Protein
Gene
How does a gene specify a polypeptide? (DNA)
Central Dogma of Molecular Biology:
Transcription
Genes code for the sequence of
nucleotides in RNA molecules.
RNA brings about formation of a mRNA
protein coded for by DNA of a
particular gene.
Protein
Cells are governed by a cellular chain of command:
DNA → RNA → protein
Central dogma
Transcription
Produces a transcript (RNA copy) of a gene
This messenger RNA (mRNA) specifies the amino acid sequence of a polypeptide
Translation
Process of synthesizing specific polypeptide on a ribosome using the mRNA
template
Transcription
A gene is an organized unit of DNA
sequences that enables a segment of
DNA to be transcribed into RNA and
ultimately results in the formation of a
functional product
Over 90% of all genes are structural
Other genes code for the RNA itself
Transfer RNA (tRNA): translates
mRNA into amino acids
Ribosomal RNA (rRNA): part of
ribosomes
Transcription occurs in 3 stages
1. Initiation
2. Elongation
3. Termination
4
The three stages of prokaryotic
transcription
1. Initiation
• Recognition step
• In bacteria, sigma factor causes RNA polymerase to recognize promoter region
• Stage completed when DNA strands separate near promoter to form open complex
2. Elongation
• RNA polymerase synthesizes RNA
• Template or noncoding strand used for RNA synthesis. Coding strand is not used
• Synthesized 5’ to 3’
• Uracil substituted for thymine
3. Termination
• RNA polymerase
reaches termination
sequence
• Causes both the
polymerase and
newly-made RNA
transcript to dissociate
from DNA
Direction of transcription
Direction of transcription and DNA strand used varies among genes
In all cases, synthesis of RNA transcript is 5’ to 3’ and DNA template strand reads
3’ to 5’
Eukaryotic transcription
◼ Basic features identical to prokaryotes
◼ However, each step has more proteins
◼ Three forms of RNA polymerase
RNA polymerase II – transcribes mRNA
RNA polymerase I – transcribes nonstructural genes for rRNA
RNA polymerase III – transcribes nonstructural genes for tRNA and 5S
rRNA
◼ RNA polymerase II requires 5 general transcription factors to initiate
transcription
RNA processing
◼ Bacterial mRNAs can be translated
immediately
◼ Eukaryotic mRNAs are made in a longer
pre-mRNA form that requires
processing into mature mRNA
Introns – transcribed but not
translated
Exons – coding sequence found in
mature mRNA
◼ Splicing – removal of introns and
connection of exons
◼ In most cases, mRNA splicing is done
by a large ribonucleoproteins complex
called “spliceosome”
◼ Other modifications – addition of tails
and caps 8
RNA processing: 5’ cap and poly A tail
◼ 5’ capping
Modified guanosine (7-methyl guanosine) attached to 5’ end
Needed for mRNA to exit nucleus and bind ribosome
◼ 3’ poly A tail
100-200 adenine nucleotides added to 3’ end
Increases stability and lifespan in cytosol
Not encoded in gene sequence
Translation
◼ Genetic code – sequence of bases in
an mRNA molecule
◼ Read in groups of three
nonoverlapping nucleotide base
triplets or codons
◼ Most codons specify a particular
amino acid. Also,
1 Start (AUG) and
3 Stop codons (UGA, UAA and
UAG)
◼ Genetic code is degenerate – more
than one codon can specify the same
amino acid
Translation of bacterial mRNA
◼ Bacterial mRNA has 5’ ribosomal-binding site
(RBS)
◼ Translation starts usually at the start codon
(AUG)
◼ Typical polypeptide is a few hundred amino
acids in length
◼ Stop codons (aka Termination or nonsense
codons)
UAA, UAG or UGA
11
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
Transcription
◼ DNA sequence of gene transcribed
into mRNA
◼ mRNA
Codon – set of 3 RNA
nucleotides
T of DNA substituted for U of
RNA
◼ tRNA
Anticodon – 3 RNA nucleotide
part of tRNA molecule
Allows binding of tRNA to
mRNA codon
tRNA
◼ Different tRNA molecules encoded by different
genes
◼ tRNASer carries serine
◼ Common features
Cloverleaf structure
Anticodon
Acceptor stem for amino acid binding
14
Aminoacyl tRNA synthetase
◼ Catalyzes attachment of amino acids to tRNA
One for each of 20 different amino acids
◼ Reactions result in tRNA with amino acid attached (charged tRNA or aminoacyl
tRNA)
◼ Ability of aminoacyl-tRNA synthetase to recognize appropriate tRNA has been
called the “second genetic code”
15
SmartBook page 257
Ribosomes
◼ Prokaryotes have one kind of ribosome.
◼ Eukaryotes have distinct ribosomes in different cellular compartments
(cytosol, mitochondria, chloroplast)
◼ Composed of large and small subunits
◼ Structural differences between prokaryotes and
eukaryotes exploited by antibiotics to inhibit bacterial
ribosomes only
◼ Overall ribosome shape determined by rRNA
◼ Discrete sites for tRNA binding and polypeptide synthesis
A site – Aminoacyl site
P site – Peptidyl site
E site – Exit site
SmartBook page 259
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 the completed polypeptide
17
Initiation of translation
◼ mRNA, the first tRNA and ribosomal subunits
assemble
◼ Requires help of ribosomal initiation factors
◼ Also requires input of energy (GTP hydrolysis)
◼ Initiation in bacteria
mRNA binds to small ribosomal subunit facilitated
by ribosomal-binding sequence
Start codon is 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
◼ Two eukaryotic differences in initiation
Instead of a ribosomal-binding sequence, mRNAs
have guanosine cap at 5’ end
◼ Recognized by cap-binding proteins
Position of start codon more variable
◼ In many cases, first AUG codon used as start
codon
Elongation of translation
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
2. 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
Called the peptidyl transfer reaction
rRNA catalyzes peptide bond formation (the
ribosome is a ribozyme)
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 site 19
Uncharged tRNA exits from E site
Termination of translation
◼ When a stop codon is found in the A site, translation
ends
◼ 3 stop codons – UAA, UAG, UGA
◼ Recognized by release factors
◼ Completed polypeptide is 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
20
Directionality
❑ 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