1
Q
Template strand
A
- The strand that is transcribed
- RNA is built from its complementary base pairs
- Read in a 3’ to 5’ direction
2
Q
Coding strand (non-template)
A
- Identical sequence to the produced RNA (except for uracil)
3
Q
In which direction is a new RNA molecule synthesized?
A
5’ to 3’ direction (new nucleotides added to the 3’ end of the strand)
4
Q
What is a requirement in the formation of phosphodiester bonds?
A
- A nucleoside triphosphate monomer
- 2 P required for energy input to form bond
- Nucleoside monophosphate becomes part of polymer
5
Q
Which protein complex transcribes DNA?
A
RNA polymerase
6
Q
Modifications to the Central Dogma
A
- Many genes do not code for proteins
Examples: - miRNA (regulation of gene transcription)
- tRNA (AA transport)
- rRNA (catalyze peptide bond formation)
7
Q
E. coli is the model organism for…
A
DNA replication, gene transcription, translation
8
Q
Prokaryotic RNA Polymerase
A
- Large globular enzyme w/ several channels
- Active site at intersection of channels
9
Q
Holoenzyme
A
- Component of prokaryotic RNApol
- Core enzyme + sigma factor
- Synthesize RNA + regulatory subunit (sigma factor)
- Core RNApol combines w/ sigma factor –> RNApol holoenzyme
10
Q
Promoter
A
- Sequence directly upstream of start of gene
- Region on non-template stand, 40-50bp long
- RNApol must recognize it + bind firmly
11
Q
Sigma factor
A
- Recognizes promoter sequence
- Positions DNA for correct transcription
- Uses -35 and -10 box sequences to position itself on non-template strand
- Most bacteria have several types of sigma proteins (E coli –> 7 types)
- Each sigma binds to promoters w/ slightly different sequences
12
Q
When does transcription begin? (pro)
A
- Starts at +1
- When sigma identifies + binds to -10 and -35 boxes, properly orienting RNApol holoenzyme for transcription at start site
13
Q
Steps of initiation + elongation (transcription in bacteria)
A
- Sigma factor binds to RNApol
- Sigma factor bind to promoter region
- Double helix of DNA is unwound (comp. strands broken apart)
- RNA synthesis begins
- Sigma factor released
14
Q
Termination (transcription in bacteria)
A
- RNApol reaches transcription termination sequence in DNA template
- Term. sequence codes for RNA to fold in on itself (hairpin) –> disrupts transcription complex (destabilizes –> falls apart)
- RNApol releases RNA transcript + DNA template
15
Q
Which strand is used as the template strand?
A
- Promoters are asymmetric –> binds RNApol in only one direction
- Depends on gene
- RNApol binds to a promoter (specifies the non-template strand) –> transcription of the template stand
16
Q
Transcription in eukaryotes vs prokaryotes
A
- Euks have DNA tightly packed + wrapped in histones (DNA packaging)
- Euk RNApol –> 3 types (vs one)
- Many euk promoters are more diverse+ complex (RNApol II –> TATA box, RNApol I + II w/ a diff set of promoters)
- Euk RNApols require large team of accessory proteins (general transcription factors assemble at promoter w/ RNApol)
- mRNA is processed before export from nucleus
- Euk genes spread out w/ gaps of 100,000 bps of untranscribed DNA between them (allows complex regulation by regulatory sequences throughout genome)
17
Q
Chromatin
A
DNA + protein (histone)
- DNA molecules combine w/ proteins –> higher order structure
- Allows for compact packaging + strict regulation of gene expression
18
Q
In what form does DNA spend most of its time?
A
Chromosome in extended form
19
Q
Initiation of transcription (euk)
A
- TATA box recognized by TBP (subunit of TFIID)
- Binding of TFIID distorts helix (kink), allowing other factors (TFIIA/B/C/etc) to pile on to form transcription initiation complex
- TFIIH pries apart double helix at transcription start point
- Once transcription starts, most of transcription factor team members come off (can help at another site) –> rNTPs come in, polymerization starts, TFIID stays
20
Q
TATA binding protein
A
Subunit of TFIID that recognizes + binds to TATA box within promoter –> causes kinks and partial unwinding of double helix
21
Q
Processing of euk mRNAs in nucleus
A
- Capping, splicing, polyadenylation –> mature mRNA
- Done by enzymes that ride on RNApol II
- Required before export from nucleus
22
Q
Capping
A
- Guanine + 3P + methyl group (7mG + 3P)
- 5’ cap
- Recognition signal for translation machinery
23
Q
Polyadenylation
A
- Poly-A tail
- 150-250 As
- Stabilizes msg
- Protects from degradation (extends 1/2 life)
- Newest part of strand
24
Q
Exons vs introns
A
- Exons: “express”, coding sequences for euk genes, will be expressed –> dictate peptide sequence
- Introns: need to be removed, non-coding sequences
25
Organization of pro vs euk genes
- Pro: continuous sequence
- Euk: exons (coding) interrupted by introns (non-coding)
26
Removing introns
- After capping, while still being transcribed, RNA splicing begins
- Each intron contains a few short sequences at/near ends --> cues for removal
27
Structure of introns
- Branch points A 'attacks' 5' splice site --> cuts sugar-phosphate backbone
- Cut end forms covalent bond w/ ribose sugar group
- Lariat (branched structure formed from intron) eventually degraded
28
Splicing
- Carried out by spliceosomes (RNA-protein complexes from non-coding RNA) --> consists of 5 small nuclear ribonucleic particles (snRNPs)
- RNA + 100+ proteins
- Catalytic activity provided by RNA component
- 'Ribozymes'
- Bring intron loop together, catalyze joining + breaking
29
Advantages of RNA splicing
- Can create diff. proteins from same gene/same primary mRNA transcript depending on cell type, stage development, gender, etc. --> 'splice variants'
- Depending on cell type: choose which exons to keep
30
Disadvantages of RNA splicing
- More steps --> more work
- More steps --> more opportunity for error
- Mutations of splice sites can result in: loss of exons, inclusion of introns, shift in locations of splice
31
Mature mRNA export from nucleus
- Cap + poly-A tail 'marker' by proteins
- EJC (exon junction complex (proteins)) binds to properly spliced mRNAs
- Only then mRNA transported out of nuclear pore into cytoplasm
32
Genetic Code
- Relationship b/w sequence of nucleotides in DNA/RNA and sequence of amino acids in protein
- 64 (4^3) possible codons, only 20 AAs
- 1 codon NEVER specifies more than 1 AA
- Almost universal (start and stop codons are the same in majority of genes in animals, plants, and microbes) --> exceptions: some fungi + protozoa, mitochondria (animal)
33
What amino acids are specified by only one codon?
Met (methionine) and Trp (tryptophan)
34
Exceptions to genetic code
- Assign some of the three STOP codons to an amino acid
Mitochondria (animal cells):
- Use UGA to encode tryptophan (instead of stop)
- Implications for transferring of mitochondrial genes to nuclear genome
- Cytosolic protein-synthesizing machinery reading mitochondrial gene will always STOP when it should be inserting Trp
Plant mitochondria (use universal code --> not an issue)
35
Reading frame mutations
- Loss or gain of bases (deletions/insertions) that shift reading frame (frame shift mutation) can lead to novel beneficial/disastrous proteins
36
How does mRNA codon specify an amino acid?
- Francis Crick: 'adapter molecule' held AAs in place while interacting directly + specifically w/ codon in mRNA
- Adapter --> tRNA
- tRNA + AA --> aminoacyl tRNA (covalent bond catalyzed by aminoacyl tRNA synthetase)
- Each AA has its own aminoacyl tRNA synthetase
37
Structure of tRNA
- 3' end --> binding site for AA
- Anticodon loop --> 3 ribonucleotides that base pair with mRNA codon
- Cloverleaf shape (hydrogen bonding b/w complementary base pairs creating loops)
- L-shape, 90 degree bend
38
Redundancy in genetic code + tRNAs
- Several different codons can specify the same AA
Specificity of tRNAs:
- Some AAs have more than 1 tRNA
- Some tRNA need accurate base-pairing at only first 2 based of a codon (can tolerate mismatch ('wobble') at 3rd position)
39
Wobble hypothesis
anticodon of tRNAs can still bind successfully to codon whose 3rd position requires a nonstandard base pairing
40
Loading tRNA w/ AA: aminoacyl-tRNA synthetase
- Total of 20 aminoacyl-tRNA synthetases (each AA has its own) --> must recognize its AA + all anticodons that recognize that AA
- Hydrolysis of ATP (spent) couples to attachment of AA to tRNA
- Combined action of tRNA + synthetases ensures each mRNA codon is matched to correct AA
41
'Charging' of tRNA
- Active site binds ATP (hydrolysis --> AMP) + AA
- AA bound to AMP is now 'activated'
- activated AA is transferred to tRNA (moves onto enzyme) from aminoacyl-tRNA synthetase (checked in 2 places: receiving end for AA, anticodon loop)
- AMP + enzyme separate from tRNA (ready for translation)
42
Components of ribosome
- Large subunit: catalyzes formation of peptide bonds (euk: 3 RNA + 39 proteins)
- Small subunit: matches tRNA to codons, initiates translation (euk: 1 RNA + 33 proteins)
- Made of RNA + proteins
- Larger than largest proteins
- Large + small subunits free in cytosol until they come across mRNA that needs to be translated
43
When does translation of a particular codon begin and end?
- Begins: anticodon of 'charged' tRNA binds to codon in mRNA
- End: AA forms peptide bond with growing chain
44
4 steps of translation cycle
1. New AA diffuses into A (acceptor) site (if codon in mRNA doesn't match with anticodon in tRNA, AA diffuses out)
2. If match, P site AA-tRNA bond breaks + peptide bond forms between P site and A site AAs
3. Large subunit moves 1 triplet to the right to move severed tRNA to E site + empty A site
4. Small subunit moves 1 triplet to the right to align with large subunit and mRNA, E site tRNA exits
Cycle is ready to repeat
45
What initiates translation?
- Always begins with AUG codon
- Initiator tRNA always loaded with met (formyl-met in bacteria)
- Initiator tRNA binds tightly to P site (only indicator tRNA can do this) along with translation initiation factors
- Small subunit w/ bound initiator tRNA, moves along mRNA searching for first AUG
- AUG found, translation initiation factors dissociate, large subunit binds
- Translation cycle begins with 1st AA (w/ tRNA) binding to A site
- All new proteins start with Methionine (typically snipped off in a later step)
46
What terminates translation?
- Presence of several STOP codons in mRNA (not recognized by a tRNA; do not specify an AA
- Any STOP codon that reaches A site will bind a release factor --> alters catalytic activity, causing addition of a water, rather than forming a peptide bond
- Frees the carboxyl end of the peptide chain --> peptide, mRNA, large + small subunits released
47
Is the ribosome an enzyme or a ribozyme (is catalytic activity provided by RNA or protein compartments)?
- A,P, E sites --> primarily RNA
- Catalytic site (where peptide bond is formed) between P and A sites in large subunit is entirely RNA
- Ribosomal proteins are mostly superficial (helping to create + maintain shape of RNA core)
48
RNA world hypothesis
- RNA did it all, including catalyzing rxns
(RNA predates DNA as a molecule)
49
Polyribosomes (polysomes)
- Many ribosomes translating the same mRNA at the same time
- Takes from 50 sec to 1-2 mins for a single ribosome to translate a protein
- Greatly increases output (as soon as first ribosome out of way)
- In both bacteria + eukaryotes
50
Simultaneous transcription + translation in prokaryotes
- No nucleus --> transcription + translation in same location
- Can greatly increase output
- Translation of mRNA while transcription of DNA is still happening
- Can have polyribosomes (polysomes) translating the mRNA
51
When do proteins fold?
- Begins during translation, long before termination + disassembly of ribosomes
- Although it doesn't require energy (spontaneous), often assisted by proteins called molecular chaperones
- Some chaperone proteins bind to ribosome near 'tunnel' where growing peptide exits
52
What is post-translational modification (PTM)?
- Chemical modifications of protein structure (20 AAs)
- Addition of functional groups or small molecules
- Major effects on charge, shape, activity
53
What are some types of post-transcriptional modifications (PTMs)?
- Glycosylation (addition of CHO)
- Lipid addition
- Phosphorylation
- Ubiquitination
- Methylation, hydroxylation, acetylation
- proteolysis (cleavage of peptide bonds)
Over 30+ types
54
How do PTMs increase protein diversity?
- Increase proteome complexity
- So many different combinations of PTMs that can be done on a single protein, let alone all of the proteins a cell can make
55
Regulation of protein production in euk cells
- Nucleus: control of protein amount (initiation of transcription)
- Cytosol: stabilization of mRNA (poly-A tail) to prevent degradation
- Degradation of fully folded proteins in cytosol
Is more being made than degraded?
