Transcript
Molecular Biology Chapter 17
Central Dogma
DNARNAProteins
Transcription of DNA to mRNA
RNA processing if you’re a eukaryote
Translation of mRNA to polypeptides
Relationship[ between nucleus and the rest of the cell
Acetbularia acetabulum
Nucleus is located down in the “foot”, can be removed without killing the cell
How will the cell be affected?
If the nucleus is where proteins are made, it should die quickly
Without a nucleus, cells can live for a few months
Proteins are still made for about 2 weeks
Transcription
Taking the information in DNA out to the rest of the cell
Controls which proteins get made
Similar rules to DNA synthesis
Bubble need to open up to access the information
NTPs (the basic building blocks) use complimentary base pairing
Only one strand is “read”
Strand read by RNA polymerase= template
Strand not read by RNA polymerase= non-template or coding strand
DNA and RNA
5’ - CGGCTCGAACTGCTA - 3’
3’ - GCCGAGCTTGACGAT - 5’ (template strand)
AFTER TRANSCRIPTION
3’ – GCCGAGCTTGACGAT – 5’
5’ – CGGCUCGAACUGCUA – 3’
RNA polymerase
Synthesizes in the 5’ – 3’ direction
Unlike DNA polymerase, does not need a primer to start
2 subunits:
RNA polymerase (core enzyme)
Sigma
RNA polymerase contains the active site
RNA polymerase won’t work without sigma
Sigma recognizes specific region in the DNA called promoter
In prokaryotes, promoters are upstream of the gene
10 bases upstream (-10) = TATAAT
Prokaryotes also have “TTGACA” at -35
Sequence can vary outside those boxes
Prokaryotic transcription- INITIATION
Sigma binds to the DNA at the promoter
More than 1 type of sigma
E.coli has 7
Streptomyces coelicolor has 60
Why have different versions of sigma?
Prokaryotic transcription- INITIATION and ELONGATION
Structure of RNA polymerase has multiple channels
DNA passage
NTP’s entrance
mRNA’s exit
Prokaryotic transcription- INITIATION
Double helix is opened up
Template is fed to the active site
NTPs enter via a channel
Prokaryotic transcription- INITIATION and ELONGATION
DNA is read 3’ – 5’. mRNA is synthesized 5’- 3’
Growing strand extends out of the RNA polymerase
About 5 bases/ second
Prokaryotic transcription- TERMINATION
Transcription ends when RNA polymerase hits a signal in the DNA
Termination sequence
Termination sequence forms a hairpin loop
RNA now ready as template for proteins
Eukaryotic transcription
Eukaryotic promoters are more varied
Generally, “TATAAA” between -25 and -35
TATA box
Also, not just 1 protein to start transcription
Lots of basal transcription factors
More than 1 RNA polymerase
RNA pol II makes mRNA
Termination occurs at a consensus sequence
Can be s f bases past the gene
Eukaryotic transcription- TERMINATION
In eukaryotes, mRNA is “immature”, a primary transcript
Eukaryotic RNAs
Eukaryotes have lots of non-coding DNA
What would be the sensible/efficient way to organize coding and non-coding DNA?
RNA processing
Regions in genes that become part of the final mRNA = exons (expressed information)
Regions in genes that don’t become part of the final mRNA = introns (intervening sequence)
Discovering introns
mRNA should bind with its template DNA due to complimentary base pairing
Test: denature double stranded DNA, allow to anneal with mRNA
In eukaryotes, mRNA matches the template, but not continuously
Genes occur in pieces!!!
Information occurs in fragments
ISRB IVZVT SPVZRING BRZCXZXVEAK YVVZET?
ISRB IVZVT SPVZRING BRZCXZXVEAK YVVZET?
Post editing
IS IT SPRING BREAK YET?
RNA processing- splicing
Have to get the introns out!
Facilitated by small nuclear ribonucleoproteins (snRNPs)
Complex of proteins and small RNA molecules
RNA acting as an enzyme
snRNPs bind to the intron
5’ end is GU, 4; end is an A
Spliceosome is formed
Intron is looped on itself, U is connected to the A
Exon fragments are ligated together
Post transcription modification
5’ cap
A molecule of 7- methyl- guanylate with 3 phosphate groups is attached to the 5’ end of the mRNA
Poly- A tail
The 3’ end of the mRNA is chopped off
Why?
Another enzyme adds on a long run of A nucleotides
About 100- 250 total
Why modify?
mRNA is more stable
End up producing more proteins
Recognition signals
Translation
mRNA to proteins
occurs at the ribosomes
controls rate of protein synthesis
in bacteria, translation starts before the mRNA is fully synthesized
Polyribosome = multiple ribosomes on a single mRNA
Polyribosomes
What traits do prokaryotes have (or don’t have) that allow them to do this?
Translation- Eukaryotes
Transcription and translation are separated by space and time
Information in the genetic code is organized into triplet codons
Translation
How does mRNA interact with amino acids?
Interact directly
Difficult to accomplish
Too much chemical variation in amino acids
Intermediary molecule
“Adapter” mole holes amino acids while interacting with the individual codons in the mRNA (Crick)
Transfer RNAs (tRNA)
Discovered by accident
“Extra” RNA was found to be required for protein synthesis in vitro
When radioactivity labeled amino acids were in the mixture, they would end up attached to this “extra” RNA, then transferred to new polypeptides
tRNAs match amino acids with codons
key properties
energy is required to attach the amino acid (charge the tRNA)
Enzymes= aminoacyl tRNA synthetases
Each amino acid has its own aminoacyl tRNA synthetase
tRNA structure
small molecules
75-85 bases long
Several stems and loops
3’ end: site of amino acid binding
Anticodon on the loop furthest from the 3’ end
Anticodon matches the codon on the mRNA
Anticodon is antiparallel to the mRNA
Tertiary structure
Structure is highly conserved between the different tRNA sequences
Distance between anticodon and the amino acid
Translation
61 different codons
But most cells only have about 30-40 different tRNAs
Wobble hypothesis (Crick)
Most amino acids have more than 1 codon
Does it matter that CCG is read as if it were CCA?
No it does not!!!
3rd position bases may not follow strict base pairing rules
Non-standard pairing are ok as long as it doesn’t change the amino acid that is specified by the codon
Ribosome (bacterial)
Ribosomes are combinations of proteins and RNAs
2 subunits
Small (30S) subunit
21 different proteins
1 16S rRNA (ribosomal RNA) molecules
Large (50S) subunit
34 different proteins
2 rRNA moles, 5S and 23S
Active site is made of RNA- ribozyme
Small subunit hold the mRNA
Large subunit synthesizes polypeptides
Suring translation, 3 tRNA molecules are held in the ribosome
A site- tRNA with amino acid
P site- tRNA holding polypeptide chain
E site- tRNA with nothing attached, ready to exit
Proteins have direction too
Synthesized from N-terminus to C-terminus
20 amino acids / second in bacteria
2/second in eukaryotes
3 phases
Initiation
Elongation
Termination
Translation- Initiation
Upstream of the start codon on the mRNA is the ribosome binding site (Shine-Dalgarno sequence)
5’ – AGGAGGU – 3’
Binds to rRNA in the small subunit with complementary sequences
tRNA with methionine binds to the start codon
Proteins called initiation factors help out
Large subunits attaches
tRNA with methionine is in the P-site
Translation begins
Translation- Elongation
Charged tRNA enters the ribosome at the A site
Which amino acid?
Peptide bond forms
Amino acid in the P site is transferred to the amino acid in the A site
Translocation
Amino acid in the P site shifts to the E site
Amino acid in the A site shifts to the P site
New charged tRNA enters the ribosome at the A site
Which amino acid?
Peptide bond forms
Amino acid in the P site is transferred to the amino acid in the A site
Translocation
Amino acid in the P site shifts to the E site
Amino acid in the A site shifts to the P site
Translation- Termination
Proceeds down the length of the mRNA until the stop codon is reached
No tRNA that matches
Instead, a release factor enters the A site
Release factor is a protein that mimics the structure of a tRNA
Does not carry an amino acid
Growing polypeptide is cut from the tRNA in the P site, but there is no attachment point in the A site
Polypeptide is released
2 remaining tRNAs are released
Ribosome separates into subunits
Can reform around the new mRNA
Translation
Are the proteins finished at this point?
Proper folding (molecular chaperones)
Chemical modifications
Where is this happening?