Download Fig. 8.1. Amino acid structure

Chapter 8
Gene Expression
taking genetic information and
using it to produce phenotypic traits
Central Dogma
transcription
translation
DNA
mRNA
template
strand
modification
protein
gene
product
phenotype
Chapter 8
8.1
Gene Expression
Polypeptide chains are linear polymers of amino acids.
proteins:
catalyzing reactions (enzymes)
regulating gene expression (regulatory proteins)
structural proteins
one or more chains of amino acids (20)
linked by peptide bonds
polypeptide chains
Chapter 8
8.1
Gene Expression
Polypeptide chains are linear polymers of amino acids.
amino acids
 carbon
carboxyl group
amino group
side chain
-COOH
-NH2
-R
connected to each other between
carboxyl group and amino group
(dehydration synthesis)
Fig. 8.1. Amino acid structure
© 2006 Jones and Bartlett Publishers
Fig. 8.2. Chemical structures of amino acids specific in the genetic code
© 2006 Jones and Bartlett Publishers
Fig. 8.3. Properties of a polypeptide chain
© 2006 Jones and Bartlett Publishers
Chapter 8
8.1
Gene Expression
Polypeptide chains are linear polymers of amino acids.
protein folding
interactions between amino acids
folding to give 3-D structure
domains
picture of beta chain of hemoglobin
showing folding/domains
Chapter 8
8.1
Gene Expression
Polypeptide chains are linear polymers of amino acids.
protein folding
interactions between amino acids
folding to give 3-D structure
domains
some proteins are made of multiple chains
each one being a subunit
picture of hemoglobin
Chapter 8
8.1
Gene Expression
Polypeptide chains are linear polymers of amino acids.
domain observations
only 7% of human proteins/domains
are specific to vertebrates
complexity is
~1.8 x fly or worm
~5.8 x yeast
vertebrate genomes have few protein domains
not found in other organisms…
…but they are more complex because they have
put them together in more complex ways
Chapter 8
8.2
Gene Expression
linear order of amino acids is encoded in the DNA.
most genes code of a single polypeptide (protein)
order of nucleotides determines order of amino acids
genes and proteins are colinear
Fig. 8.4. Colinearity of DNA and protein in the trpA gene of E. coli
© 2006 Jones and Bartlett Publishers
Chapter 8
8.1
8.2
Gene Expression
REVIEW
Polypeptide chains are linear polymers of amino acids.
linear order of amino acids is encoded in the DNA.
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
synthesis of RNA is similar to that of DNA
•RNA is made from single stranded DNA
•monomers are ribonucleotides A, C, G and U
Fig. 8.5. Structural differences between ribose and deoxyribose and between
uracil and thymine
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
synthesis of RNA is similar to that of DNA
•RNA is made from single stranded DNA
•monomers are ribonucleotides A, C, G and U
•sequence of bases is determined by DNA sequence
•nucleotides connected 5’-P to 3’-OH
•nucleotides only added at the 3’ end of RNA
•enzyme is different - RNA polymerase(s)
can initiate without a primer
Fig. 8.6A, B. RNA synthesis
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
RNA polymerases
prokaryotes - RNA polymerase holoenzyme
six polypeptide chains
can process more than 104 nucleotides
(while associated with the template)
processivity
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
RNA polymerases
eukaryotes
- larger, more subunits
RNA polymerase I
RNA polymerase II
RNA polymerase III
makes rRNA
mRNA, snRNA’s, processing
tRNA’s, 5S rRNA
processivity > 106 nucleotides
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
which strand
where to start
where to stop
?
•promoter recognition
•chain initiation
•chain elongation
•chain termination
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
•promoter recognition
which strand
where to start
RNA polymerase binds to promoter
regions of DNA,
20-200 bp
“recognized” by RNA polymerase
consensus sequences (see fig. 8.8)
*
TATA box
binding strength varies
~ closer to consensus has stronger binding
(Eukaryotes also have enhancers that interact with promoters)
Fig. 8.8. Base sequences in promoter regions of several genes in E. coli
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
•promoter recognition
•chain initiation
after RNA polymerase binding
transcription begins at +1
only one strand is transcribed
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
•promoter recognition
•chain initiation
•chain elongation
next nucleotide added to 3’ end
RNA made in 5’ to 3’ direction
about 17 bp of DNA are separated
double helix reforms
RNA trails off as separate strand
Fig. 8.6C. RNA synthesis
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
•promoter recognition
•chain initiation
•chain elongation
•chain termination
special DNA sequences
RNA polymerase dissociates from DNA
self termination
sequence only
which strand ?
RNA sequence ?
RNA polymerase
terminates transcription
when loop forms in
transcript
Fig. 8.9. (A) Base sequence of a transcription termination region; (B) the 3' terminus
of an RNA transcript
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
Fig. 8.10. EM of part of newt DNA showing tandem repeats
of genes . [Courtesy of Oscar Miller and Barbara R. Beatty]
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
•promoter recognition
•chain initiation
•chain elongation
•chain termination
special DNA sequences
RNA polymerase dissociates from DNA
self termination
sequence only
termination protein
sequence and protein
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
•promoter recognition
•chain initiation
•chain elongation
•chain termination
mutations
in coding region
in promotor
in termination sequence
change amino acids
no transcript ?
long transcript ?
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
Transcription
only one strand is transcribed
might be either one
(either strand can have promoters/terminators)
genes usually don’t overlap
A
B
C
Fig. 8.11. Typical arrangement of promoters and termination sites in a segment of a
DNA molecule
© 2006 Jones and Bartlett Publishers
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
RNA transcript is called the primary( 1°) transcript
mRNA
5’
5’ untranslated region
3’
open reading frame
(ORF)
3’ untranslated region
Chapter 8
8.3
Gene Expression
DNA sequence determines RNA sequence.
RNA transcript is called the primary( 1°) transcript
in prokaryotes:
used as mRNA directly for protein synthesis
short lifetime (minutes)
in eukaryotes:
primary transcript is processed to become mRNA
longer lifetime (hours to days)
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA processing
1. terminal cap is added
at 5’ end
add modified guanosine
5’ to 5’ linkage
needed for mRNA to bind to ribosome
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA processing
1. terminal cap is added
2. poly-A tail is added
add up to 200 A to the 3’ end
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA processing
1. terminal cap is added
2. poly-A tail is added
3. remove introns
take out unnecessary RNA
resplice needed RNA
exon
5’
intron
exon
intron
exon
3’
Fig. 8.12. mRNA processing in eukaryotes
© 2006 Jones and Bartlett Publishers
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA processing
Many steps involved in processing are coupled
For example:
proteins involved with RNA polymerase to promote
elongation also help recruit splicing machinery
the splicing machinery helps to:
speed up elongation
recruit the polyadenylation machinery
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing (in the nucleus)
takes place at spliceosomes
nuclear particles
protein and small RNA’s
forming snRNP’s
U1, U2, U4, U5, U6
small
nuclear
ribonucleoprotein
particles
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing (in the nucleus)
5 snRNP RNA:
U1, U2, U4, U5, U6
U4 and U6 are normally paired, U2 is stable alone
U1 binds to both ends of the intron
and brings them together
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing (in the nucleus)
U2 destabilizes U4-U6 complex
and displaces U4 (U2 binds to U6)
U2 also binds to 3’ end of intron
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing (in the nucleus)
U1, U2, U4, U5, U6
U4 and U6 are normally paired, U2 is stable alone
Fig. 8.13 . Interactions between small nuclear RNAs in snRNPs that
are involved in splicing
© 2006 Jones and Bartlett Publishers
Fig. 8.14B. Drawing of DNA-RNA hybrid
© 2006 Jones and Bartlett Publishers
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing
hybridize DNA with processed RNA
(denature / renature)
mRNA
DNA
Chapter 8
8.3
8.4
Gene Expression
DNA sequence determines RNA sequence.
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing (in other places)
mitochondria
Tetrahymena
happens w/out spliceosomes
self slicing RNA
ribozymes
titin has 178
typical is about 87 bp
Table 8.2. Characteristics of human
genes
BRAC1 has 21 introns
spread over 100,000 b
mRNA = 7800 b
peptide has 1863 a.a.
© 2006 Jones and Bartlett Publishers
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing
human genes are spread out
have small exons separated
by long introns
only about 5% of a gene codes for protein
longest human gene is muscle protein, dystrophin
2.4 Mb (79 exons)
codes for over 3500 amino acids
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
RNA splicing
many exons correspond to domains
of the assembled protein
suggests that some current genes
may have been assembled from
smaller pieces
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
many genes
?
more proteins
a single primary transcript can be spliced in different
ways to give different mRNA (thus different proteins)
sxl-protein
http://fig.cox.miami.edu/~cmallery/150/gene/split_genes.htm
non-functional
protein
+
Chapter 8
8.4
Gene Expression
Eukaryotic 1° transcript is processed to become mRNA
tropomyosin
http://departments.oxy.edu/biology/Stillman/bi221/111300/processing_of_hnrnas.htm
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
protein production includes two processes:
information transfer
getting the amino acids in the correct order
chemical synthesis
hooking the amino acids together
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
protein production has 5 major components
•mRNA
- needed for assembly of ribosome
has information for amino acid sequence
•ribosomes
- 2 subunits, align tRNA’s, attach a.a.’s
•tRNA
- carry appropriate amino acid, have anticodon
•aminoacyl-tRNA synthetases
•factors
- puts a.a.’s on tRNA
- for initiation, elongation and termination
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Overview:
initiation
elongation
termination
mRNA binds to ribosome
tRNA’s are brought in one by one with a.a.
adjacent amino acids are joined
finished protein is released from ribosome
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Eukaryotic initiation:
eIF
=
eukaryotic Initiation Factors
not elongation factors (pg. 294)
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Eukaryotic initiation:
eIF4F binds to 5’ cap of mRNA
recruits eIF4A and eIF4B
Fig. 8.15. Initiation of
protein synthesis
© 2006 Jones and Bartlett Publishers
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Eukaryotic initiation:
eIF4F binds to 5’ cap of mRNA
recruits eIF4A and eIF4B
creates binding site for:
eIF2, eIF3, eIF5, tRNAMet
small 40S subunit of ribosome
making initiation complex 48S
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Eukaryotic initiation:
eIF4F binds to 5’ cap of mRNA
recruits eIF4A and eIF4B
creates binding site for:
eIF2, eIF3, eIF5, tRNAMet
small 40S subunit of ribosome
making initiation complex 48S
scans for AUG
eiF5 causes release of initiation factors
and recruitment of the 60S subunit
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Ribosome (60S subunit) has three binding sites
E
Exit
P Peptidyl
A
Aminoacyl
Fig. 8.15. Initiation of
protein synthesis
hydrogen bonding
between codon
and anticodon
© 2006 Jones and Bartlett Publishers
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Elongation (three steps)
•bring in next tRNA (with amino acid)
•form new peptide bond
•move to next codon on mRNA
Energy for elongation is provided by:
EF-2 - GTP
EF-1 - GTP
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Elongation
1. 40S subunit shifts one codon “down” the message
new “charged” tRNA is brought to A site
2. coupled reaction forms new peptide bond
(peptidyl transferase activity)
3. large subunit moves to “catch up” to small subunit
tRNA’s are shifted
1. from P and E site
1. to the A and P site
1
1
2
3
Fig. 8.16A, B. Elongation cycle in protein synthesis
3
© 2006 Jones and Bartlett Publishers
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Elongation
completed one cycle
repeat for next codon
1
1
2
3
Fig. 8.16C, D. Elongation cycle in protein synthesis
3
© 2006 Jones and Bartlett Publishers
Fig. 8.16. Elongation cycle in protein synthesis
© 2006 Jones and Bartlett Publishers
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Elongation
eukaryotes
prokaryotes
40S
60S
12-15 aa/sec
30S
50S
20 aa/sec
EF-1
EF-2
EF-Tu
EF-G
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Terrmination (release phase)
eukaryotic termination codons:
UAG
UAA
UGA
RF
prokaryotes
UAA
RF-1
UAG
UAA
RF-2
UGA
Fig. 8.18. Termination of protein synthesis
© 2006 Jones and Bartlett Publishers
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
Initiation
Elongation
Termination
protein folding
most proteins fold as they are being synthesized
aa with hydrophilic R
aa with hydrophobic R
-helix
-pleated sheet
surface
internal
Chapter 8
Gene Expression
alpha helix
O
H
http://wiz2.pharm.wayne.edu/biochem/nsphelix1.jpg
Chapter 8
Gene Expression
beta pleated sheet
http://www.sciencecollege.co.uk/SC/biochemicals/bsheet.gif
Fig. 8.19. A "ribbon" diagram of the path of the backbone of a polypeptide.
[Adapted from W. I. Weiss, et al. 1992. Nature 360: 127.]
© 2006 Jones and Bartlett Publishers
Fig. 8.20. Alternative
pathways in protein
folding
© 2006 Jones and Bartlett Publishers
Chapter 8
8.5
Gene Expression
Translation takes place on a ribosome
eukaryotic
one protein / mRNA
reads from 5’cap - to
termination codon
prokaryotic
may be polycistronic
(multiple proteins / mRNA)
can initiate in other areas
AGGAGG
(Shine-Dalgarno sequence)
for example, 10 enzymes needed for histidine synthesis - one mRNA
Fig. 8.21. Products translated from a three-cistron mRNA molecule
© 2006 Jones and Bartlett Publishers
write from
by convention: DNA
protein
L to R
5’ to 3’
amino to carboxyl
Fig. 8.22. Direction of synthesis of RNA and of protein
© 2006 Jones and Bartlett Publishers
Chapter 8
8.6
Gene Expression
Genetic code for amino acids is a triplet code
list of all codons and amino acids they encode
4 =4
4x4 = 16
4x4x4 =64
codons are linear and non-overlapping
Fig. 8.23. Reading bases in an RNA molecule
© 2006 Jones and Bartlett Publishers
reading frame
frameshift mutation
Fig. 8.24. Change in an amino acid sequence of a protein caused by the addition of an
extra base
© 2006 Jones and Bartlett Publishers
Fig. 8.25. Interpretation of the rll frameshift mutations
© 2006 Jones and Bartlett Publishers
Chapter 8
8.6
Gene Expression
Genetic code for amino acids is a triplet code
make synthetic polynucleotides
AAAAAAAAAAAA…
Lys Lys Lys Lys…
UUUUUUUUUUUU… Phe Phe Phe Phe…
CCCCCCCCCCCC… Pro Pro Pro Pro…
GGGGGGGGGGGG… Gly Gly Gly Gly…
translate in vitro and look at peptides made
Fig. 8.26. Polypeptide synthesis in three different reading frames
© 2006 Jones and Bartlett Publishers
redundancy
more than one way to
get most amino acids
universality (almost)
minor differences in
some protozoans
some organelles
Table 8.3. The standard genetic code
© 2006 Jones and Bartlett Publishers
Chapter 8
8.6
Gene Expression
Genetic code for amino acids is a triplet code
tRNAs
(how many different ones?)
small, single stranded RNA
70-90 nucleotides long
5’ is monophosphate (instead of triphosphate)
folds on itself
anticodon region
3’ end for attachment of a.a.
2-D
Fig. 8.27. tRNA cloverleaf configuration
© 2006 Jones and Bartlett Publishers
“wobble” at the third position
number of distinct tRNAs is
less than the # of codons
3’
5’
3-D
Fig. 8.28B. Diagram of the three-dimensional structure of yeast tRNAPhe
© 2006 Jones and Bartlett Publishers
Table 8.4. Wobble rules for tRNAs of
E. coli and S. cervisiae
© 2006 Jones and Bartlett Publishers
Chapter 8
8.7
Gene Expression
Multiple ribosomes can move in tandem on mRNA
After ribosome has moves about 75 nucleotides another
ribosome can initiate translation on the same message
in prokaryotes (no nucleus)
can have simultaneous transcription and translation
Chapter 8
8.7
Gene Expression
Multiple ribosomes can move in tandem on mRNA
http://www.phschool.com/science/biology_place/biocoach/images/translation/polysome.gif