Lecture 13 Flashcards
1
Q
What is the relationship between DNA, RNA and proteins?
A
DNA makes RNA makes protein
2
Q
Where does transcription and translation fit in?
A
DNA -> RNA (transcription)
RNA -> proteins (translation)
3
Q
Where does transcription occur? Where does translation occur?
A
- Transcription occurs in the nucleus
- Translation occurs in the cytoplasm
4
Q
Where does transcription initiation, transcription termination and translation initiation and translation termination occur?
A
SEE SEPERATE SHEET FOR THIS - REALLY GOOD AND CLEAR DIAGRAM (sheet with PINK STAR on)
red line = transcription start site
red cross = transcription end site
blue line = translation start site
blue cross = transcription end site
promoter - includes the TATA box
blue box in the diagram are the untranslated region
- Transcription
- Capping and tailing
- Splicing
- Translation
5
Q
What is a gene?
A
Codes for one functional unit:
• either a sequence of amino acids in a polypeptide
• or a sequence of nucleotides in an untranslated RNA
6
Q
Types of RNA
A
- mRNA - messenger RNA
- rRNA - ribosomal RNA
- tRNA - transfer RNA
Other types too:
• noncoding RNA
• snRNA - small nuclear RNA
• miRNA - microRNA
• antisense RNA
7
Q
RNA versus DNA (what are there differences)
A
- RNA has C2 hydroxyl group
- RNA contains U instead of T
- RNA molecules are single-stranded
- RNA often forms stemloops: complementary, antiparallel strands, which form a (mini) helix
8
Q
How does the RNA pentose sugar differ from DNA pentose sugar?
A
9
Q
What does RNA stemloops look like?
A
RNA often forms stemloops: complementary, antiparallel strands, which form a (mini) helix
10
Q
RNA has complex 3D DNA
A
Circled picture shows the base forming a structure together
11
Q
Ribosomal RNA (rRNA)
A
- >80% of total RNA
- few kinds, many copies
- small and large rRNAs • highly conserved
- Prokaryotes: 1x small, 2x large (e.g.16S rRNA)
- Eukaryotes: 2x small, 2x large (e.g.18S rRNA)
12
Q
tRNA
A
- >15% of total RNA
- ~100 kinds, many copies
- small (79 nt)
- each tRNA is dedicated (cognate) to one of 20 amino acids
13
Q
mRNA
A
- ~ 2-5% of total RNA
- 100.000s kinds, few copies
- ‘mRNA transcripts’
14
Q
What do you need to be able to do?
A
- Describe in broad terms ‘What is a gene?’
- Explain how ‘the code carries the function’
- Describe the main differences between DNA and RNA
- Explain how RNA can form complex 3D structures
- Describe the three main types of RNA: ribosomal RNA, transfer RNA and messenger RNA
- Appreciate that there are many other types of RNA, with important functions in the cell
15
Q
Define gene expression
A
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
16
Q
Gene expression processes require:
A
- need for a template
- need for an enzyme (and lots of other factors…)
- need for substrates
17
Q
What are the three steps in transcription?
A
- initiation
- elongation
- termination
18
Q
Recap from other lectures, what is the template, enzymes and substrates in DNA replication?
A
- template: DNA
- enzyme: DNA polymerase
- substrates: dNTPs
19
Q
Transcription - Making RNA (what is the template, enzymes, substrates?)
A
- template: DNA
- enzyme: RNA polymerase
- substrates: NTPs
20
Q
What is actually happening in transcription (formula, enzymes, direction of synthesis…)
A
(RNA)n + NTP <-> (RNA)n+1 + PPI
- catalysed by RNA polymerase
- hydrolysis of PPi by pyrophosphatase drives reaction
- double-stranded DNA template
- NTPs and Mg2+ needed
- synthesis from 5’ to 3’
21
Q
Which bonds are breaking etc in transcription?
A
22
Q
Main types of RNA (which enzymes forms these RNA?)
A
- rRNA - ribosomal RNA, made by RNA polymerase I
- mRNA - messenger RNA, made by RNA polymerase II
- tRNA - transfer RNA, made by RNA polymerase III
23
Q
RNA polymerase (describe the inactive form and the active form)
A
- 1 copy: RNAP = bacterial RNA polymerase
- Core enzyme has 5 subunits, the core enzyme is the inactive enzyme
- Holoenzyme has 6 subunits: core enzyme + σ (sigma) - the holoenzyme is the active enzyme
24
Q
What is the structure of the RNAP (RNA polymerase) core enzyme?
A
Not active in it’s 5 subunit form
25
RNA polymerase holoenzyme
Sigma (σ) subunit needed for specific DNA binding on promoter
The holoenzyme is now active as it has the sigma subunit bound
26
What does 'transcription is ubiquitous' mean?
It means it happens like this everywhere in the body
27
Transcription initiation
- TATA box (short sequence of DNA, part of the promoter region) is recognised by a transcription factor
- Transcription factors bind to the promoter
- Binding is directional on the TATA box
- Transcription factors also bind to upstream sequences, the way the transcription facotrs bind to promoters, controls when a gene is expressed and when a gene is not expressed.
- RNA polymerase is recruited
(Transcription inititation complex - the transcription factors and RNA polymerase. The complex initiates transcription and RNA polymerase begins RNA synthesis by matching up the complementary bases to the original DNA strand)
- unwinding DNA helix
- formation of a 'transcription bubble' (17 base pairs)
- transcription initiates, directionally 5' to 3' (RNA polymerase starts catalysising the reaction to make RNA)
28
Transcription elongation
* RNA synthesis from 5’ to 3’
* template strand is read 3’ to 5’
* single-stranded RNA molecule is made
* only one DNA strand is used as template (as it is a single stranded molecule made)
* as transcription bubble advances, DNA helix re-forms behind it
29
Names of the DNA strands
• DNA template strand is transcribed
o complementary to the new RNA strand
o complementary to ‘the other DNA strand’
• non-transcribed DNA strand is coding strand
• transcribed DNA strand is non-coding strand
30
Transcription termination
* Sequence-dependent, other factors involved
* Results in a primary RNA molecule
* RNA processing needed to produce mature RNA
31
What is RNA processing? (what happens after rRNA and tRNA are transcribed, what happens after mRNA is transcribed)
- post-transcriptional modifications
What happens after rRNA and tRNA have been transcribed?
- Chemical modifications and cleavage (to make the complex 3D structures)
What happens after mRNA is transcribed?
- Eukaryotes: 5' capping (affects the 5' end), 3' tailing (affects the 3' end), splicing (affects the middle)
32
RNA processing - capping
* 5’ Cap
* Methylated-G linked 5'-5'
* Provides protection
* Plays role in translation
33
RNA processing - tailing
* 3’ poly A tail
* for protection
* for regulation
- A cleavage signal is being recognised 'AAUAAA'
- Endonuclease cuts slightly down stream of this cleavage signal
- The extra bit of RNA made after the cleavage signalling will then be degraded
- Addition of tail of A's put on to the cleavage signal. This is done by the enzyme poly (A) polymerase
34
RNA processing - splicing
- highly accurate removal of introns (leaves exons only)
- removal of introns, exons joined together
• by spliceosomes
o ribonucleoprotein complex (snRNAs+proteins)
• mature mRNA contains ORF (open reading frame) plus 5’UTR and 3’UTR (untranslated region)
• splicing errors can cause disease
35
Outcomes - what do you need to be able to do?
* Describe the general outline of the mechanism of transcription initiation, elongation and termination
* Appreciate how binding of Transcription Factors initiates transcription, and thereby control gene expression regulation in eukaryotes
* Define coding strand and template strand, and explain which one is transcribed
* Outline the processes involved in RNA processing: capping, tailing and splicing
36
Ribosomes (structure)
Eukaryotes
• 4 rRNAs + lots of proteins (can see the rRNA strands)
• 40S (smaller subunit) + 60S (larger subunit) subunits
• 80S ribosome
- can see the polypeptide coming out of the exit tunnel in the ribosome
37
Genetic code (features of this)
* from 4-letter ‘DNA language’ to 20-letter ‘protein-language’
* adaptor molecule needed: tRNA
* triplet code, degenerate (single amino acid may be coded for by more than one codon)
* non-overlapping and ‘comma-less’ (no gaps)
* 5’ to 3’ template read-through producing N to C polypeptide chain extension
38
What is the translation initiation codon?
Initation codon: AUG (Met)
Termination codons: UAA, UAG, UGA
39
Structure of tRNA (talk about how the 'right' tRNA binds, with the 'right' amino acid)
* the ‘genetic adaptor’
* each tRNA is dedicated (cognate) to one of 20 amino acids
* each tRNA has an anticodon to recognise a complementary codon in the mRNA
40
tRNA charging (what is this? what enzymes are involved?)
1. Aminoacyl-tRNA synthetase (enzyme) binds to a specific amino acids
2. ATP then 'charges up' the amino acid
3. This same aminoacyl-tRNA synthetase enzyme will recognise a specific tRNA using the anticodon on the tRNA, it will bind the specific amino acid to the specific tRNA.
4. This tRNA is now chraged (as it has an amino acid attached)
For different tRNA's, that need to be loaded up with different amino acids, a different enzyme is needed.
41
What three 'main' things are needed in translation, what other things are also needed in translation?
* mature mRNA
* ribosomes
* tRNAs charged with activated amino acids
In addition…
• initiation factors (IFs), elongation factors (EFs), release factors (RFs) and energy (ATP/GTP)
Must learn all of this slide
42
Explain what happens in initiation (translation)
1. Mature mRNA
Cap is recognised by: CAP binding proteins, initiation factors, 40S subunit, tRNA\*Met (this means the tRNA is bound to the Met), GTP
2 and 3. The 'construct' is made and moves along the mRNA to find the AUG (energy is required for this)
4. The 60S subunit is then recruited, and this leads to a functional ribosome formed. The functional ribosome is now ready, it has the tRNA with the Met bound to the AUG on the mRNA.
43
Summary of translation - initiation
* Recognition of cap by tRNA\*-Met
* Recognition of the AUG by anticodon of ‘initiation complex’
* Initiation Factors (IFs) involved
* Recruitment of large ribosomal subunit (60S)
* Assembly of functional ribosome
Now we have:
• ‘80S initiation complex’ with tRNA\*-Met in P-site on AUG on mRNA, and empty A-site, ready for elongation… (A is ready = ready to recieve the next triplet code)
44
What does EPA 5' to 3' mean?
45
Explain what happens in elongation in translation
* binding of aminoacyl-tRNA on A-site
* peptide bond formation (between the two amino acids) catalysed by peptidyltransferase
* translocation, ribosome moves one codon, from 5’ to 3’
* uncharged tRNA in E-site
* growing polypeptide ‘pushed’ into exit tunnel
* Elongation Factors (EFs) involved
* peptidyl-tRNA in P-site and empty A-site, ready for next elongation cycle…
(don't worry about the E side - this isn't shown in the diagram)
46
Where is the exit tunnel?
Located in the large sub-unit in the ribosome
47
How many sites of the EPA is occupied at one time?
• peptidyl-tRNA in P-site and empty A-site, ready for next elongation cycle…
• only 2 sites (of EPA) occupied at any one time as it moves along the mRNA
48
Termination (in translation)
* peptidyl-tRNA in P-site and empty A-site with stopcodon
* RF recognises stop codon
* binding of Release Factor (RF) bringing 1 molecule of water. The release factor looks like tRNA, but it isn't, it is a protein.
* This water causes the hydrolysis reaction, resulting in releasing the free peptide chain (through tunnel) and uncharged tRNA in P-site
* Dissociation of ribosome into subunits
49
Outcome - What do I need to be able to do?
* Describe the general structure of ribosomes in prokaryotes and eukaryotes
* Describe the general structure of transfer RNA and explain how it functions as the genetic adaptor molecule
* Explain the principles of the genetic code, and be confident using the genetic code
* Describe the general outline of the mechanism of translation initiation, elongation and termination
