1
Q
Central Dogma of (Molecular) Biology
A
- Three aspects: information storage, information carrier, active cell machinery
- Flow of genetic information from DNA to RNA (transcription), RNA to protein (translation)
o Segments of DNA that are transcribed into RNA are called genes - DNA can be copied or replicated to produce new DNA molecules
2
Q
Differences/similarities between RNA and DNA
A
- RNA contains the sugar ribose, DNA contains deoxyribose (presence of an additional -OH group)
- RNA contains the base uracil, the equivalent base in DNA is thymine (absence of a -CH3 group)
- The chemical linkage between nucleotides in RNA and DNA is a phosphodiester bond
3
Q
Transcription from a DNA Template
A
- Only one strand of DNA is transcribed – template strand
o In a 5’ to 3’ direction - Other DNA strand called non-template (coding) strand
o Sequence of ‘coding’ DNA strand matches RNA, except U in place of T
4
Q
Formation of Phosphodiester Bonds:
A
- result of two hydroxyl groups in phosphoric acid reacting with hydroxyl groups on other molecules to form two ester bonds – forming the backbone of nucleic acids
- In DNA and RNA specifically, the phosphodiester bond is the linkage between the 3’ carbon atom of one sugar molecule and the 5’ carbon atom of another, Deoxyribose in DNA and ribose in RNA
o Strong covalent bonds form between the phosphate group and two 5-carbon ring carbohydrates (pentoses) over two ester bonds
5
Q
Modifications to Central Dogma
A
- Many genes code for RNA molecules that are not mRNA – do not code for proteins
- These RNAs are involved in: regulation of gene transcription (miRNA), processing of mRNA prior to translation (spliceosome), translation – transport of amino acids (tRNA) and catalyze the formation of peptide bonds (rRNA)
6
Q
mRNAs code for
A
proteins
7
Q
rRNAs form
A
the core of the ribosome and catalyze protein synthesis
8
Q
miRNAs
A
regulate gene expression
9
Q
tRNAs
A
serve as adaptors between mRNA and amino acids during protein synthesis
10
Q
other small RNAs
A
used in RNA splicing, telomere maintenance, and many other processes
11
Q
- e. coli
A
DNA replication, gene transcription, translation
12
Q
- saccharomyces cerevisiae (yeast)
A
cell cycle “minimal model eukaryote”
13
Q
- Arabidopsis thaliana
A
all flowering plants closely related
14
Q
- Drosophila melanogaster
A
genetics, development
15
Q
- C. elegans (“the worm”
A
– first animal genome to be sequenced; location, lineage and fate of every cell in embryo, larva, and adult is known
16
Q
- Mouse
A
‘model mammal’ genetics well understood
17
Q
Prokaryotic RNA polymerase
A
- Large, globular enzyme with several channels running through it
- Active site is at the intersection of these channels
- Holoenzyme is made up of the core enzyme, which can synthesize RNA and regulatory subunit (sigma factor)
18
Q
Prokaryotic RNA polymerase pt 2
A
- RNA polymerase must be able to recognize start of a gene and bind firmly to DNA at this site
o Promoter – sequence immediately upstream of start of gene
19
Q
sigma factor
A
recognizes a promoter sequence
* Transcription initiated at specific sections of DNA called promoters
o Regions on non-template strand, 40-50 bp long
* Most bacteria have several types of sigma proteins (e.g. e coli has 7 types)
o Each sigma binds to promoters with slightly different sequence
20
Q
Prokaryotic Promoters and Initiation of Transcription
A
- 10 box – 10 bases upstream from start site
- 35 box – 35 bases upstream (transcription starts at +1)
- Typical sequences are found at boxes, rest of promoter is highly variable
- Transcription begins when sigma identifies and binds to -10 and -35 boxes, properly orienting the RNA polymerase holoenzyme for transcription at start site
21
Q
Transcription in Bacteria: Initiation & Elongation
A
- Sigma opens helix; transcription begins, building blocks (NTP) added
Transcription in Bacteria: termination - Polymerase reaches transcription termination signal in DNA template
o Codes for RNA that folds back on itself, forming hairpin structure that disrupts transcription complex
Polymerase releases RNA transcript and DNA template
22
Q
transcription in bacteria recap
A
- Starting 3’ to 5’ on template strand, RNA polymerase will recognize promoter and RNA synthesis will begin
- Sigma factor released and polymerase clamps firmly down on DNA and RNA synthesis will continue
- As RNA transcript grows, the termination and release of both polymerase and completed RNA transcript
23
Q
what strand is used as the template?
A
- Promoters are asymmetric -> binds polymerase in only one direction
- Which strand? – depends on gene, polymerase binds to promoter specfies on non-template strand, transcription of template strand
24
Q
Transcription in Eukaryotes (vs. prokaryotes)
A
- eukaryotes need to deal with DNA packaging
- wrapped around histones, tightly packed
- eukaryotes have three distinct types of RNApol (vs one)
- RNApol I, II, III
- eukaryotic promoters are more diverse and complex
- many promoters recognized by RNApol II include a sequence called a TATA box (30 bp upstream)
25
Transcription in Eukaryotes (vs. prokaryotes) pt 2
* RNApol I and III interact with completely different sets of promoters
* eukaryotic RNApols require large team of accessory proteins
* general transcription factors must assemble at promoter, along with RNApol
* mRNA undergoes processing before being exported from nucleus
* eukaryotic genes can be spread out, with gaps of up to 100,000 bps of untranscribed DNA between them
* allows for complex regulation of gene transcription by regulatory sequences scattered through genome
26
Higher Order DNA Structure
* DNA molecules combine with proteins to adopt higher order structure
* Allows for compact packaging and strict regulation of gene expression
27
Eukaryotic RNA polymerases
* RNApol I – transcribes most rRNA genes
* RNApol II – protein-coding genes, miRNA genes, plus genes for some small RNAs (e.g. those in spliceosomes)
* RNApol III – tRNA genes, 5S rRNA gene, genes for many other small RNAs
28
Initiation of Transcription in Eukaryotes
* TATA box recognized by TBP (TATA binding protein), a subunit of TFIID
* binding of TFIID distorts helix, allows other factors (TFIIA, B, C, etc) to pile on to form ‘transcription initiation complex’
* TFIIH pries apart double helix at transcription start point …
29
TATA binding protein
* subunit of TFIID that recognizes and binds to TATA box within promoter
* causes kinks and partial unwinding of double helix
* core ‘TATA’ sequence: 5’-TATAAA-3’
30
Processing of Eukaryotic mRNAs in the Nucleus
* various modifications required before transcripts can be exported from nucleus
* capping, splicing, polyadenylation
* carried out by enzymes that ride on RNA polymerase II
31
mRNA capping and polydenylation
- 5' cap is the recognition signal for translation machinery
- poly-a-tail protects 3' end from degradation
32
Organization of Prokaryotic vs Eukaryotic Genes
coding sequences of eukaryotic genes (exons) interrupted by non-coding sequences (introns)
- in bacterial gene, no interruptions
33
Eukaryotic DNA: Coding & Non-Coding Regions
- to make functional mRNA, entire gene (introns + exons) transcribed
- primary transcript or pre-mRNA
- after capping, while still being transcribed, RNA splicing (removal of introns) begins
- each intron contains a few short sequences at/near its ends that are cues for its removal
34
Each intron forms branched structure (lariat)
- branch point A ‘attacks’ 5’ splice site -> cuts sugar-phosphate backbone
- cut end forms covalent bond with ribose sugar group
- lariat structure eventually degraded
35
Splicing is carried out by RNA-protein complexes.
spliceosomes:
consist of 5 small nuclear ribonucleic particles (RNA-protein complex) (snRNPs, pronounced ‘snurps’) RNA + 100+ proteins
- catalytic activity provided by RNA component
‘ribozymes’
36
How many exons in a ‘typical’ eukaryotic gene?
2000 nucleotide pairs in a human beta-globin
- codes for β-chain of hemoglobin; mutated in sickle cell anemia, beta thalassemia, …
- 200,000 nucleotide pairs -- clotting factor deficient in hemophilia A
37
Advantages of RNA Splicing
- can create different proteins from same gene / same primary mRNA transcript depending on cell type, stage development, gender, etc. ‘splice variants’
37
Advantages of RNA Splicing pt 2
- α- tropomyosin interacts with actin, expressed in all cell types
- different isoforms have different functions
- muscle cells -> regulation of contraction
- other cell types -> involved in microfilament functions
38
Disadvantages of RNA Splicing
- more steps -> more work!
- more steps means more opportunity for error
- mutations of splice sites can result in loss of exons, inclusion of introns, shift in location of splice
39
Only mature mRNA is exported from nucleus.
- cap and poly-A tail are ‘marked’ by proteins
- group of proteins called exon junction complex (EJC) binds to properly spliced mRNAs
- only then will mRNA be transported out of the nuclear pore into cytoplasm
BIOL 130 Midterm
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