Transcript Molecular Biology review

What is the flow of information
through the cell?
Double helix - antiparallel polymers
Major groove
Minor groove
5’
3’
A
Purine
G
T
Pyrimidine
C
06_12_asymmetrical.jpg
Transcription:
• dsDNA template
• Nucleotides (ACGU)
make ssRNA
• Need to separate
strands.
• Nucleotides added to
free 3’ OH (5’3’)
Classes of RNA…
mRNA
rRNA
…also snRNA and microRNA
tRNA
Prokaryotes
untranslated regions
5’ UTR
promoter
(not transcribed)
3’ UTR
coding
sequence
DNA
mRNA
RNA Polymerase
Ribosome
polypeptide
Eukaryotes
untranslated regions
5’ URT
promoter
(not transcribed)
3’ UTR
coding
sequence
DNA
pre mRNA
mRNA
RNA Polymerase
Ribosome
polypeptide
Bacterial Promoter Elements
•
•
•
•
Transcription start = +1
Consensus sequence = –35; TTGACA, recognized by 
Pribnow box = -10, TATAAT; determines +1
Terminator sequence: where polymerase stops
Initiation of transcription in prokaryotes
Initiation of eukaryotic
transcription by RNA Pol
II (mRNA)
TF = transcription factor
(compare with prokaryotic
sigma factor)
Eukaryotic
mRNA:
• 5’ cap,
• 5’ UTR
• coding region
• 3’ UTR
• 3’ poly-A tail
mRNA processing
in Eukaryotes
5’ cap added
Remove 3’ end
Poly-A tail added
Introns removed
Exons joined
Gene cloning - making lots of copies...
1.
2.
3.
4.
Make a “library” of small pieces of DNA (2 types)
Find the one piece you want
Insert it into a “vector”
Grow it in a new organism (bacteria, euk. cells)
replication
transcription
DNA
translation
RNA
Isolate DNA,
fragment
with RE
Genomic
library
Protein
Isolate mRNA, convert
to cDNA with reverse
transcriptase
cDNA
library
Overview of
gene expression
in eukaryotes
07_37_Protein.produc.jpg
07_28_ribosome.jpg
Two adapters link an amino acid to a codon
07_26_2_adaptors.jpg
Intiation of
translation in
Eukaryotes
07_32_initiation.jpg
Intiation of
translation in
Prokaryotes
07_33_mRNA.encode.jpg
Elongation of
proteins
07_30_3_step_cycle.jpg
4
5
07_34_stop codon.jpg
Termination of
translation
Mutations…
• Frameshift: Adding or removing 1 or 2 nucleotides results
in changes the reading frame from that point on.
• Nonsense: Changing an amino acid codon to a stop codon
results in truncated proteins
• Missense: Changing an amino acid codon to one encoding
a different amino acid - effect depends on type of amino
acid and where in the protein.
04_03_20 amino acids.jpg
Side chains
interact via
all of the
noncovalent
bonds
Primary structure (1°) of a protein:
Arabidopsis -glucosidase (single letter codes)
MSSLHWFPNIFIVVVVFFSLRSSQVVLEEEESTVVGYGYVVRSVGVDSNRQVLTAKLDLIKPSSV
YAPDIKSLNLHVSLETSERLRIRITDSSQQRWEIPETVIPRAGNHSPRRFSTEEDGGNSPENNFL
ADPSSDLVFTLHNTTPFGFSVSRRSSGDILFDTSPDSSDSNTYFIFKDQFLQLSSALPENRSNLY
GIGEHTKRSFRLIPGETMTLWNADTGSENPDVNLYGSHPFYMDVRGSKGNEEAGTTHGVLLLNSN
GMDVKYEGHRITYNVIGGVIDLYVFAGPSPEMVMNQYTELIGRPAPMPYWSFGFHQCRYGYKNVS
DLEYVVDGYAKAGIPLEVMWTDIDYMDGYKDFTLDPVNFPEDKMQSFVDTLHKNGQKYVLILDPG
IGVDSSYGTYNRGMEADVFIKRNGEPYLGEVWPGKVYFPDFLNPAAATFWSNEIKMFQEILPLDG
LWIDMNELSNFITSPLSSGSSLDDPPYKINNSGDKRPINNKTVPATSIHFGNISEYDAHNLYGLL
EAKATHQAVVDITGKRPFILSRSTFVSSGKYTAHWTGDNAAKWEDLAYSIPGILNFGLFGIPMVG
ADICGFSHDTTEELCRRWIQLGAFYPFARDHSSLGTARQELYLWDSVASSARKVLGLRMRLLPHL
YTLMYEAHVSGNPIARPLFFSFPQDTKTYEIDSQFLIGKSIMVSPALKQGAVAVDAYFPAGNWFD
LFNYSFAVGGDSGKHVRLDTPADHVNVHVREGSIVAMQGEALTTRDARKTPYQLLVVASRLENIS
GELFLDDGENLRMGAGGGNRDWTLVKFRCYVTGKSVVLRSEVVNPEYASKMKWSIGKVTFVGFEN
VENVKTYEVRTSERLRSPRISLIKTVSDNDDPRFLSVEVSKLSLLVGKKFEMRLRLT
Secondary
structure (2°)
-helix
H-bonds
between
C=O and N-H of
backbone.
(No R-groups
involved)
Secondary
structure
-sheet
H-bonds
between
C=O and N-H
of backbone.
(No R-groups
involved)
Tertiary structure - the entire
polypeptide
-helix
-sheet
loops and turns
disulfide bridge
ribonuclease
Quaternary
structure multiple subunits
e.g. hemoglobin
cytochrome b562
lactic (lactate)
dehydrogenase
immunoglobulin
light chain
04_20_protein domains.jpg
Domains - discrete
modules within
tertiary structure
that fold
independently and
have a specific
function.
4 domains of
phospholipase C
Motif - a recurring substructure
/ barrel
e.g. -amylase
Motif - a recurring substructure
coiled coil
e.g. myosin
How do proteins get to their folded state?
unfolded
native
conformation
Proteins with different functions may have similar shape members of a family with a common ancestor.
04_21_Serine proteases.jpg
04_22_protein subunit.jpg