Transcript MBP 1022h Lecture 4_Chapt 6

Chapter 6
• How Cells Read the
Genome: From DNA to
Protein
– RNA
– Transcription
– RNA processing
– Translation
RNA contains Ribose and Uracil
DNA contains Deoxyribose and Thymine
Phosphodiester bonds between RNA and DNA are the same
RNA is single stranded, thus it can fold into specific
structures by base-pairing with complementary sequences
TRANSCRIPTION:
RNA Polymerase
Recognize initiation site
Separation of DNA
Base pairing
Energy requiring process
No primer required
Process begins at specific
site within the promoter
Promoter
RNA polymerase:
DNA is transcribed by RNA polymerase
No primer required
Small window is made in DNA approximately 9 bases in length
MOVIE
RNA polymerase can move in either direction
RNA polymerase uses the antisense strand as a template
to produce primary RNA strand
Genes can be expressed with different efficiencies
RNA polymerase requires a protein complex at
a promoter to initiate RNA synthesis
Enhancer binding proteins can act from a distance to
enhance initiation of transcription.
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
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.
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
Prokaryotes
(operon)
Diff chromosomes; one mRNA
Eukaryotes
Trp mRNA
5’
3’
Proteins
Polycistronic
Monocistronic
Eukaryotes: Organization of DNA within a single gene; Exons
and introns
Primary transcript
Exon
Intron
Exon
Intron
Intron
Exon
Intron
Primary transcript
Exon
modification
Exon Exon
Splicing
Cap
poly A tail
RNA PROCESSING
The structure of two human genes
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
Alternative splicing
The primary transcript can be spliced into
several distinct mRNAs
Spliced and polyA mRNA is exported through the nuclear pore
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.
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)
The genetic code
Three different reading frames
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 > a.a. ; tRNA can attach to
more than one codon (basis for wobble in genetic code)
How do you charge a tRNA with an aa
Several different views of a tRNA
The wobble position is found at
the third base of the codon
Polypeptide synthesis is a processive reaction
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
The ribosome is a ribonucleoprotein
mRNA Translation
Step: 1. Basepair to codon, A site
2. A new peptide is formed
3. Ejecting spent tRNA and
resetting for next round
MOVIES
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' -> 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.
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 aminoacyltRNA; 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.
movie
DNA to Protein
Each step in the synthesis of a protein can ultimately
affect the amount of protein found in a cell