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BIT 220 Chapter 12
Basic process of
Transcription
Figures 12.3
Figure 12.5
Terminology
Molecular Biology/Genetics: the study of
gene structure and function at the molecular
level
Molecular Biotechnology: the ability to
transfer specific units of genetic
information from one organism to another
Recombinant DNA Technology
= gene splicing
= genetic engineering
=gene transplantation
= gene cloning
=molecular cloning
DNA
Measured in base pairs (bp)
kb - kilobases - 1000 bases
10.4 base pairs in one helical turn
3.4A (3.4 x 10-4 um) -space between each base pair
Molecular Weight of 1 NT pair 660
Genes are conventionally written with
non-template (coding) strand on top
in 5’-3’ direction
Template in 3’-5’ direction
Genes
Definition
•Linear sequence of DNA that contains all the
necessary information for the production of a
protein
•promoter – start codon—gene -- stop codon
(ATG)
(introns
exons)
TERMINOLOGY
•Express Gene - Transcribe/Translate
MAKE PROTEIN
•Intron
noncoding region of DNA
•Exon
coding region of DNA
•Upstream
5’ end
•Downstream
3’ end
•ORF Open Reading Frame
from start to stop codon
Figure 12.4
Figure 12.4
DNA TRANSCRIPTION
Make single stranded complementary RNA strand
from DNA
Figure 12.7
(1) CODING STRAND of DNA=
‘identical’ to mRNA (exception uracil)
called nontemplate strand
(2) TEMPLATE
Nucleotides complementary to DNA template are
bonded to form mRNA (transcript, sense strand)
RNA as intermediary - Figure 12.8
1. Ribose Sugar
2. Phosphate
3. Four Bases
adenine
guanine
cytosine
uracil
Figure 12.9
TRANSCRIPTION BUBBLE
unwound DNA created by RNA polymerase
site of transcription process
Steps in Transcription
A. Initiation
B. Elongation
C. Termination
Figure 12.10
RNA Polymerase
•Protein
•1 prokaryotes
•3 in eukaryotes
•Holoenzyme (Complete Enzyme)
1.Core
a. alpha
recognizes upstream element
b. Betabinds ribonucleotides
forms phosphodiester bonds
c. Beta’
binds DNA template
2. Sigma recognizes the core promoter elements (-35)
directs polymerase to initiate transcription
at a specific promoter
Promoter/Regulation
Prokaryotes
•where RNA polymerase
sits on DNA and begins transcription
upstream of promoter (minus designation)
•downstream of promoter (plus designation)
•Prokaryotes
a. -10 Box
•TATA box
Eukaryotes
a. TATA box (-30)
b. CAAT box (-80)
b. -35 box
c. GC box
•b. UP element
•Figure 12.11
Initiation
1. Polymerase sits on non-specific DNA
2. Sigma subunit searches and finds promoter
3. Polymerase binds to -35 region
3. B and B’ bind tightly to DNA
4. DNA begins melting at -10 region
5. Complementary nt are added and bonded at +1
6. Sigma subunit falls off polymerase
Elongation - Figure 12.12
Transcription occurs in 5’-3’ direction
Asymmetrical
Termination of
Transcription
Figure 12.13
Intrinsic Terminators
GC rich region in DNA
Allows hairpin in RNA
•Cause polymerase to stall
•All subunits of polymerase fall apart
•mRNA is free
•DNA recoils
Transcription in
Eukaryotes
•Promoters
•Transcription Factors
•Enhancers (100-1000 bp upstream)
•Transcription in Nucleus
•Translation in Cytoplasm
•3 RNA Polymerases (Table 12.1)
Eukaryotic RNA Editing
1. 5’ capping Figure 12.19
7 methyl guanosine cap
2. polyA tail (3’) Figure 12.20
3. Introns removed - next slide
FIGURE 12.5 and 12.15
Figure 12.17 for Initiation in Eukaryotes
Intron Splicing
Figure 12.28
Most, but not all, eukaryotic genes contain introns.
These introns are removed from mature mRNA
They contain sequences which are often conserved.
MECHANISMS
(rRNA)
Self Splicing (Autocatalytic Activity)
intron excised via
two phosphoester bonds
(mRNA)
Spliceosomes
small nuclear RNA
proteins
(tRNA)
Endonuclease and Ligase
Why use RNA?
•Protect DNA
•Allows control of proteins made
•RNA needs to move to new location