BIOL 200 (Section 921)
Lecture # 4-5, June 22/23, 2006
• UNIT 4: BIOLOGICAL INFORMATION FLOW - from
DNA (gene) to RNA to protein
• Reading:
ECB (2nd Ed.) Chap 7, pp. 229-262; Chap 8, pp. 267286 (focus only on parts covered in lecture). Related
questions 7-8, 7-9, 7-11, 7-12, 7-14, 7-16, 7-17, 85a,b,d;
or
ECB (1st ed.) Chap 7, pp. 211-240; 263-4; Chap 8, pp.
257-270 (focus only on parts covered in lecture). Related
questions 7-9, 7-10, 7-12, 7-13, 7-15, 7-18, 7-19; 87b,c,e.
Lecture # 4,5: Learning objectives
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Understand the structural differences between DNA and RNA, and
the directionality of transcription.
Understand the structure of a eukaryotic gene.
Describe the general mechanism of transcription, including
binding, initiation, elongation and termination. Discuss factors
regulating transcription.
Describe processing for all three types of RNA, and discuss why it
must occur.
Describe the structural features of ribosomes.
Describe the genetic code and how it is related to the
mechanisms of transcription and translation.
Describe the coupling of tRNA and discuss the specificity of amino
acid tRNA synthetases.
Describe the general mechanism of translation including binding,
initiation, elongation and termination.
Genetic information flow [Fig. 7-1]
Transcription-same
language (nuc. to nuc.)
Translation-different languages
(nuc. to protein)
Eucaryotic vs. Procaryotic Biol. Info Flow [Fig. 7-20]
DNA vs. RNA: Differences in chemistry [Fig. 7-3]
DNA vs RNA (Lehninger et al. Biochemistry)
DNA vs. RNA: Intramolecular base pairs in RNA
[Fig. 7.5] and Intermolecular base pairs in DNA [Fig.
5-7] assist their folding into a 3D structure
RNA
DNA
Procaryotic vs. eucaryotic
transcription
[Fig. 7-12]
Fig. 7-12,
p. 219
In procaryotes, clusters of genes called operons can
be carried by one mRNA [Fig. 8-6]
Fig. 7-9:Parts of a gene [Fig. 7-9]
Promoter
sequence
terminator
DNA template
Transcription
start site
Transcription factor binding to DNA [Fig. 8-4]
What types of
bonds can you
identify between
this DNA and
protein?
Eukaryotic transcription factors promote
RNA Polymerase II binding [Equiv. Fig. 8-10, 2nd Ed.]
Directions of transcription along a short portion
of bacterial chromosome [Fig. 7-10, p. 218]
5’
3’
DNA always read 3’ to 5’!
RNA always made 5’ to 3’!
3’
5’
RNA Polymerase:
(1) Unwinds and rewinds DNA;
(2) Moves along the DNA one nucleotide at a time; (3) Catalyzes
The formation of phosphodiester bonds from the energy in the
Phosphate bonds of ATP, CTR, GTP and UTP.
Transcription by RNA polymerase in E. coli
[Lehninger et al. Principles of Biochemistry]
Fig. 7-37
preRNA
DNA in
nucleus
5’ cap
splicing
polyA tail
mature mRNA
Fig. 7-12: mRNAs in eukaryotes are
processed
Prokaryote
mRNA
Eukaryote
mRNA
5’ cap
3’ tail
Fig 7-12B
5’ cap
Functions:
- Increased stability
- Facilitate transport
- mRNA identity
Bacterial vs. Eukaryotic genes [Fig. 7-13]
RNA splicing removes introns [Fig. 7-15]
RNA splicing: (1) done by small nuclear ribonuclear protein molecules
[snRNPs, called as SNURPs] and other proteins [together called
SPLICEOSOME], (2) Specific nucleotide sequences are recognized at each
end of the intron
Fig. 7-17
RNA Splicing by
SnRNP+proteins=spliceosome
5’splice site
snRNP
3’ splice site
exon1
intron
lariat
formation
Fig. 7-17
RNA Splicing
Spliceosome
Mature mRNA
exons joined togetherintrons removed
Lariat=
loop of
intron
Exons
joined
Removal of intron in the form of a lariat [Fig. 7-16]
Detailed mechanism of RNA splicing [Fig. 7-17]
Is RNA splicing biologically
important or a wasteful process?
• Pre-mRNA can be spliced in different ways
(alternate RNA splicing), thereby generating
several different mRNAs which code for several
different proteins [e.g. approx. 30,000 human
genes can produce mRNAs coding for more than
100,000 proteins]
• Introns quickens the evolution of new and
biologically important proteins e.g. three exons of
the β-globin gene code for different structural and
functional regions (domains) of the polypeptide
Alternative splicing can produce different proteins
from the same gene [Fig. 7-18]
Becker et al. The World of the Cell
Transcription of two genes seen be EM [Fig. 7-8]
Becker et al. The World of the Cell
Biological information flow in procaryotes
and eukaryotes [Fig. 7-20]
Specialized RNA binding proteins (e.g. nuclear
transport receptor) facilitate export of mature mRNA
from nucleus to the cytoplasm [Fig. 7-19]
rRNA processing [Fig. 6-42 in Big Alberts]
45S transcript
18S
Small
ribosomal
subunit
5.8S
28S
5S made
elsewhere
Large
ribosomal
subunit
Processing of transfer RNA (tRNA) [Becker et al. The World of the Cell]
From RNA to Protein: translation of
the information
• Translation: conversion of language of 4
nucleotides in mRNA into language of 20 amino
acids in polypeptide
• Genetic code: a set of rules (triplet code) which
govern translation of the nucleotide sequence in
mRNA into the amino acid sequence of a
polypeptide
• Codon: a group of three consecutive nucleotides in
RNA which specifies one amino acid (e.g. ‘UGG’
specifies Trp and ‘AUG’ specifies Met)
Genetic code -
triplet, universal, redundant
and arbitrary [Fig. 7-21]
mRNA codons
Corresponding amino acid that will be
attached onto tRNA
mRNA can be translated in three possible reading frames
“START” and “STOP” codons on mRNA are
essential for proper mRNA translation
[Becker et al. The World of the Cell, Fig. 22-6]
Genetic code problem:
5’ AGUCUAGGCACUGA 3’
For the RNA sequence above, indicate the
amino acids that are encoded by the three
possible reading frames.
If you were told that this segment of RNA
was close to the3’ end of an mRNA that
encoded a large protein, would you know
which reading frame was used?
Frame 1-AGU CUA GGC ACU GA
Frame 2-A GUC UAG GCA CUG A
Frame 3-AG UCU AGG CAC UGA
Frame 1-Ser – Leu – Gly - Thr
What are the main players involved
in the process of translation
1. Ribosomes [“protein synthesis machines”]: carry out
the process of polypeptide synthesis
2. tRNA [“adaptors”]: : align amino acids in the correct
order along the mRNA template
3. Aminoacyl-tRNA synthetases: attach amino acids to
their appropriate tRNA molecules
4. mRNA: encode the amino acid sequence for the
polypeptide being synthesized
5. Protein factors: facilitate several steps in the
translation
6. Amino acids: required as precursors of the
polypeptide being synthesized
Ribosome Assembly
Fig. 7-28
Eukaryotes
Large=60S
Small=40S
Prokaryotes
Large=50S
Small=30S
Centrifugation-separates organelles and
macromolecules (panel 5-4)
Heavy particles move
to pellet, e.g. nucleus
Fixed angle
Swinging bucket
Svedberg unit, S, shows how fast a particle
sediments when subject to centrifugal force.
From Becker,
World of the
Cell, p. 327
Fig. 7-23: tRNA structure
Old ‘cloverleaf’
3-D L-shaped
model
model
Amino acid
attachment
H-bonds
between
complementary
bases
anticodon
Aminoacyl tRNA synthase charge-links
tRNA with amino acids [Fig 7-26]
High
Energy
Ester
bond
Fig. 7-26,
part 2
tRNA anticodon
binds mRNA
codon
Fig. 7-28
28S
Ribosomes
made of
rRNA and
protein
5.8S
5S
large
subunit
Come
together
during
translation
18S
small
subunit
Ribosomes are protein synthesis factories [Fig. 7-29]
Ribosomes can be free in the cytosol or
attached to the ER membranes
Fig. 7-35: polyribosomes or polysomes
Fig. 7-32: Initiation of translation
Start codon
Initiator rRNA-Met
+small subunit
Large subunit binds
Fig. 7-32: translation
Initiator tRNA in P
site
Aminoacyl-tRNA
binds to A site
Ribosome shift:
one codon
tRNA1 moves from
P site to E site;
tRNA2 moves from
A site to P site
Peptide
bond forms
Fig. 7-30:
Elongation of
polypeptide
Bind:
aminoacyltRNA anticodon
to mRNA codon
Bind
Bond
Bond: make
peptide bond
Shift: move ribosome
along mRNA, move
tRNAs
Shift
Fig. 7-34: termination
Stop
codon at
A site
Fig. 7-34:termination
Release factor binds
stop codon at A site
Release C terminal
from tRNA+shift
Release
ribosome
from
mRNA
Protein destruction
• Proteolysis: digestion of proteins by
hydrolyzing peptide bonds
• Proteases: enzymes that digest proteins
• Many proteases aggregate to form a
proteasome in eukaryotes
• Proteins are tagged for destruction by a
molecule called ubiquitin
• Several proteins are also degraded in
lysosomes in eukaryotes
Proteasome-mediated degradation of shortlived and unwanted proteins [Fig. 7-36]
Mechanism of ubiquitin-dependent
protein degradation
[Becker et al. The World of the Cell, Fig. 23-38]
SUMMARY
1.
2.
3.
4.
Transcription
RNA processing
Translation
Protein destruction
Transcription of two genes seen be EM [Fig. 7-8]
1.
2.
3.
4.
5.
Where are Polymerases?
Where are transcription start/stop sites?
Where are the 3’ and 5’ ends of the transcript?
Which direction are the RNA polymerases moving?
Why are the RNA transcripts so much shorter than the length
of the DNA that encodes them?