How Cells Read the Genome: From DNA to Protein

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Chapter 6

• How Cells Read the Genome From DNA to Protein
• RNA
• Transcription
• RNA processing
• Translation

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RNA contains Ribose and Uracil DNA contains
Deoxyribose and Thymine Phosphodiester bonds
between RNA and DNA are the same
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RNA is single stranded, thus it can fold into
specific structures by base-pairing with
complementary sequences
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(No Transcript)
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TRANSCRIPTION
Promoter
RNA Polymerase Recognize initiation
site Separation of DNA Base pairing Energy
requiring process No primer required Process
begins at specific site within the promoter
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RNA polymerase DNA is transcribed by RNA
polymerase No primer required Small window is
made in DNA approximately 9 bases in length
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MOVIE
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RNA polymerase can move in either direction
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RNA polymerase uses the antisense strand as a
template to produce primary RNA strand
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Genes can be expressed with different efficiencies
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RNA polymerase requires a protein complex at a
promoter to initiate RNA synthesis
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Enhancer binding proteins can act from a distance
to enhance initiation of transcription.
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• RNA Processing
• Prokaryotes transcription and translation can
  be
• concurrent.
• Eukaryotes Nucleus (RNA synth) and cytoplasm
• (Prot synth) are separated.
• Primary transcript undergoes several
  modifications.
• 5 cap is added to 5 nucleotide m7Gppp
  (Stability)
• String of adenylic acids are added to the 3 end
  (Poly
• A tail)
• Splicing internal cleavage to excise introns
  followed
• by ligation of coding exons

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5-methyl cap of mRNAs Once 25 nucleotides are
synthesized the 5 end of the primary transcript
is modified. Used to ID mRNA and protect from
degradation.
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Organization of Genes differ in prokaryotes and
eukaryotes Gene Unit of DNA that contains the
info to specificy synthesis of a single
polypeptide Compact, colinear mRNA
Diff chromosomes one mRNA
Prokaryotes (operon)
Eukaryotes
Trp mRNA
5
3
Proteins
Monocistronic
Polycistronic
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Eukaryotes Organization of DNA within a single
gene Exons and introns
Primary transcript
Exon Intron Exon Intron
Primary transcript
Exon Intron Exon Intron
modification
Exon Exon
Splicing
Cap poly A tail
RNA PROCESSING
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The structure of two human genes
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• RNA splicing
• A specific A attacks the 5 splice site
• Upon cleavage the free OH group reacts with
• the next exon sequence releasing the intron

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Alternative splicing
The primary transcript can be spliced into
several distinct mRNAs
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Spliced and polyA mRNA is exported through the
nuclear pore
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TRANSLATION

• The three roles of RNA in protein synthesis
• messenger RNA (mRNA). This will later be
  translated into a polypeptide.
• (Carries information in the form of a
  three-base code)
• transfer RNA (tRNA). RNA molecules that carry
  amino acids to the growing
• polypeptide. (Is key to deciphering the code)
• ribosomal RNA (rRNA). This will be used in the
  building of ribosomes
• machinery for synthesizing proteins by
  translating mRNA.
• (physically move along the mRNA molecule,
  catalyze assembly of a.a. into prot.

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Genetic information is carried as three base
genetic code Four bases (A G C T/U) must encode
for 20 a.a. Therefore a combination is required
43 64 Triplet code is called a CODON that must
begin at a precise site Of 64, 61 specify
individual a.a. and three are STOP codons

• starting codon is AUG (methionine)
• Code is universal, synonymous, degenerate
• Reading frame
• 3rd base in codon wobble
• frameshifts/deletions/insertions
• (MUTATIONS)

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The genetic code
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Three different reading frames
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The decoding function and process (tRNA and
aminoacyl tRNA synthases) 3D structure important
for function
The acceptor stem includes the 5' and 3' ends of
the tRNA. The 5' end is generated by RNase P. The
3' end is the site which is charged with amino
acids for translation. Some aminoacyl tRNA
synthetases interact with both the acceptor 3'
end and the anticodon when charging tRNAs.
Each tRNA is recognized by only one synthase
tRNA gt a.a. tRNA can attach to more than one
codon (basis for wobble in genetic code)
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How do you charge a tRNA with an aa
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Several different views of a tRNA
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The wobble position is found at the third base
of the codon
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Polypeptide synthesis is a processive reaction
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Ribosomes are the protein synthesizing
machinery Composed of rRNA and more than 50
proteins Small and large subunit present in
cytosol Similar 3D structure across species
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The ribosome is a ribonucleoprotein
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mRNA Translation
Step

• Basepair to codon, A site
• A new peptide is formed
• Ejecting spent tRNA and
• resetting for next round

MOVIES
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The Steps of Translation 1. Initiation The
small subunit of the ribosome binds to a site
"upstream (on the 5' side) of the start of the
message. It proceeds downstream (5' -gt 3') until
it encounters the start codon AUG. Here it is
joined by the large subunit and a special
initiator tRNA. The initiator tRNA binds to the P
site (shown in pink) on the ribosome. In
eukaryotes, initiator tRNA carries methionine
(Met). (Bacteria use a modified methionine
designated fMet.) 2. Elongation An
aminoacyl-tRNA (a tRNA covalently bound to its
amino acid) able to base pair with the next codon
on the mRNA arrives at the A site (green)
associated with an elongation factor (called
EF-Tu in bacteria) GTP (the source of the needed
energy). The preceding amino acid (Met at the
start of translation) is covalently linked to the
incoming amino acid with a peptide bond (shown in
red). The initiator tRNA is released from the P
site. The ribosome moves one codon downstream.
This shifts the more recently-arrived tRNA, with
its attached peptide, to the P site and opens the
A site for the arrival of a new aminoacyl-tRNA.
This last step is promoted by another protein
elongation factor (named EF-G) and the energy of
another molecule of GTP.
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Note the initiator tRNA is the only member of
the tRNA family that can bind directly to the P
site. The P site is so-named because, with the
exception of initiator tRNA, it binds only to a
peptidyl-tRNA molecule that is, a tRNA with the
growing peptide attached. The A site is so-named
because it binds only to the incoming
aminoacyl-tRNA that is the tRNA bringing the
next amino acid. So, for example, the tRNA that
brings Met into the interior of the polypeptide
can bind only to the A site. 3. Termination
The end of the message is marked by one or more
STOP codons (UAA, UAG, UGG). No tRNA molecules
have anticodons for STOP codons. However, a
protein release factor recognizes these codons
when they arrive at the A site. Binding of this
protein releases the polypeptide from the
ribosome. The ribosome splits into its subunits,
which can later be reassembled for another round
of protein synthesis. Polysomes A single mRNA
molecule usually has many ribosomes traveling
along it, in various stages of synthesizing the
polypeptide. This complex is called a polysome.
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movie
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DNA to Protein
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Each step in the synthesis of a protein can
ultimately affect the amount of protein found in
a cell