Download RNA Transcription

October 9
Richard Losick
Where we’ve been and where we are going!
•
DNA structure and DNA replication
Today and Next Tuesday
•
Gene transcription and the translation of RNA
into protein
Final two lectures
•
How genes are switched ON and OFF
•
How life arose on Earth, if it did arise on Earth!
Gene transcription and mRNA maturation
1. Central dogma and gene transcription
3. RNA polymerase
4. mRNA maturation in eukaryotes
Goal: To understand how genetic information is
transmitted from the genome to the ribosome.
Objectives: You should be able to:
• compare and contrast transcription with replication.
• describe the transcription machinery.
• describe how transcripts are processed into mRNAs.
2. Central Dogma and Gene Transcription
"The central dogma states that
once 'information' has passed into
protein it cannot get out again. The
transfer of information from nucleic
acid to nucleic acid, or from nucleic
acid to protein, may be possible,
but transfer from protein to protein,
or from protein to nucleic acid, is
impossible. Information means
here the precise determination of
sequence, either of bases in the
nucleic acid or of amino acid
residues in the protein.“
Francis Crick, 1958
The Central Dogma states that information
flows from nucleic acids into protein
Transcription takes place
asymmetrically in a moving bubble
(There is no “lagging” strand)
Non-template strand
?
Template strand
Is the question mark the 3’ or 5’ terminus?
3. RNA polymerase
RNA polymerase recognizes punctuation marks in
the DNA known as promoters
RNA polymerase in bacteria recognizes sequences located
10 (“-10”) and 35 (“-35”) base pairs upstream of the start site of
transcription (+1)
TTGACA and TATAAT are a Platonic ideal!
closed complex
open complex in which 14 bp of DNA are unwound
Promoter recognition is built into bacterial RNA polymerase.
initiation when the first phosphodiester bond is formed
elongation
termination
closed complex
open complex with ~13 base pairs unwound
initiation when the first phosphodiester bond is formed
elongation
termination
closed complex
open complex
initiation – formation of the first
phosphodiester bond
elongation
termination
closed complex
open complex
initiation
elongation
termination
closed complex
open complex
initiation
elongation
termination
Crab claw demonstration
Promoter recognition works differently in
eukaryotes!
Eukaryotic RNA polymerase does not recognize
promoters.
Instead, “transcription factors” recruit RNA polymerase.
The most important is TATA- binding protein (TBP).
TBP recognizes a sequence called the TATA box.
TATA-binding protein binds in the minor groove and
causes the DNA to bend.
TATA-binding protein (TBP) binds
to the TATA box.
Next, TBP recruits additional
transcription factors to the
DNA (colored shapes).
This assemblage recruits
RNA polymerase.
“Recruits” means that by diffusion RNA
polymerase bumps into the assemblage and is then
held there by binding to it.
Finally, other factors trigger
DNA melting, open complex
formation and initiation of
transcription.
Main points
1. Transcription is asymmetric and occurs with the
release of the RNA transcript.
2. Transcription is catalyzed by RNA polymerase,
which binds to promoters.
3. Bacterial promoters consist of -10 and -35
sequences.
4. In eukaryotes, transcription factors, such as the
TATA-binding protein, bind to the promoter and
recruit RNA polymerase.
4. mRNA maturation in eukaryotes
mRNA undergoes maturation events before it
exits the nucleus and is translated:
CAPing
Tailing
Splicing
Transcripts acquire a “CAP” at
their 5’ end.
The CAP is a guanine
nucleotide joined to mRNA by a
5’-5’ linkage.
3’-G-5’ppp5’-N-p........
Transcripts acquire a tail of about 200 As at their 3’
terminus.
exon
CAP
intron
exon
(A) n
Coding sequences (“exons”) in mRNA are interrupted
by non-coding sequences, “introns”, which are
removed before the mRNA is translated.
• Eukaryotic genes are in pieces.
• The coding segments are exons and the
interruptions are introns.
• The introns are removed by “splicing” after the gene
is transcribed into RNA.
Splicing occurs in two trans-esterification reactions
First, the 2’ hydroxyl of the branch point A attacks the
phosphoryl group of the G at the 5’ splice site to
create a lariat.
The lariat is a 2’ 5’ branch structure!
The 3’ OH of the 5’ exon becomes the nucleophile
in the 2nd trans-esterification reaction.
This joins the 5’ to the 3’ exon, releasing of the lariat.
To review, splicing occurs in two trans-esterification
reactions
Story time!
Joan Steitz
Translation
Goal: To understand how mRNA is translated into protein.
Objectives: You should be able to:
• describe the principal features of the genetic code.
• describe tRNA charging and how fidelity is achieved.
• describe the principal features of the ribosome.
• explain the translation cycle and how fidelity is achieved.
• explain how open-reading frames are set
The genetic code translates the 4-letter alphabet of
mRNA into the 20-word language of protein.
What is the minimum number of nucleotides needed to
specify 20 amino acids? Is it two, three, four…?
If mRNA were read in units of two nucleotides, it could
specify only 16 (42) amino acids –too few!
Ergo, messenger RNA must be read in units of (at least)
three nucleotides.
If it were read in units of the three, the number of
permutations would be 43 or 64.
The Rosetta Stone of Life!
Key features of the genetic code
Codons are three nucleotides long.
Codons are read by the ribosome in a
5’ to 3’ direction: 5’-XXX-3’.
61 triplets specify amino acids.
The remaining three are stop codons.
The Genetic Code
How is the 4-base language of mRNA
translated into the 20-amino acid language of
protein?
tRNAs are adaptors between
codons and amino acids.
Each tRNA is “charged” by a
specific amino acid and
recognizes a particular codon.
Cloverleaf Structure of tRNA
amino acid attachment at 3’ end
anticodon pairs with codon in mRNA
Tertiary Structure of tRNA
amino acids attachment at 3’ end
anticodon pairs with codon in mRNA