TOPIC III Gene Expression and translation Flashcards
The Rho protein is involved in the _____ stage of transcription.
Termination
Which of the following is correct regarding sigma factor?
It recognises the promoter sequence
Which of the following occurs first in transcription?
Formation of a haloenzyme
What is a haloenzyme?
Enzyme activated by a cofactor
Unwinding of the DNA during transcription is the result of the activity of a helicase enzyme downstream of the RNA polymerase. (True or false)
False
The RNA transcript being produced by the RNA polymerase is complementary to the template strand of the DNA. (True or False)
True
What are features that are common to all tRNA synthetases?
- Active site catalysing the covalent attachment of amino acids to tRNAs
- Binding surface for the tRNA anticodon
- Binding site for ATP
Where are promoters for Ecoli genes located?
35 bp and 10 bp upstream of first transcribed base
-35 and -10 are separated by 15 to 17 bp) - same side of double helix (important for RNA polymerase to recognise them
What are the two forms of prokaryotic RNA polymerase in initiation?
Core enzyme (5 subunits) and the Holoenzyme (core and the sigma factor subunit)
What occurs at the RNA polymerase structure in prokaryotes?
One side of sigma factor binds to -35 and -10 regions of promoter on DNA - other side forms interactions with other subunits
- DNA is unwound around -10 region and one strand fed into the channel in RNA polymerase -goes all the way though and incerperates the Mg2+ ion in active site. `
What dos the sigma factor ‘tell’ the RNA polymerase ?
Helps it to read the signals in the DNA that tell it where to begin -recognises the -35 and -10 regions
What is the function of 2 alpha subunits (protein) in RNA polymerase?
- Help with enzyme assembly, promote interactions with regulatory proteins
What is the function of the beta subunit in RNA polymerase (prokaryotes)?
Catalytic centre
What is the function of the beta prime subunit?
Binds DNA
What is the function of the omega subunit?
Enzyme assembly, REGULATION OF GENE EXPRESSION
What is the function of the sigma 70 subunit?
Binds -10 and -35 regions in promoter and helps separate the DNA strands - creates the transcription bubble by melting DNA
What is the sequence of RNA molecule the same as?
The coding strand (except for T because it is replaced by U)
Does RNA polymerase read in the 5’–> 3’ or vice verse?
It READS in 3’–> 5’ (moving right to left) and then it SYNTHESISES from 5’–>3’
Which protein is needed for the sigma factor to form an open complex with the RNA polymerase (activator protein)
The NtrC protein- binds to enhancer sequence -causes a loop to form where it can then bind to RNA polymerase to cause gene to go over promoter sequence and then SWITCHES GENE ON
When has transcription officially begun?
When the gene is SWITCHED ON
What are the two types of terminators in trsnscription ?
Intrinsic terminators and Rho dependent terminator sequence
What occurs in Intrinsic terminators?
When RNA polymerase recognises instability in the A-U bonds, the RNA sequence terminates and RNA falls off template
How come intrinsic termination just leads to RNA simply ‘falling off’ the template?
Due to the RNA strand being in a hairpin loop structure- normally it would go back and fix in linear strcutre BUT loop acts as a roadblock so it can;t go back to fix…it just drops off
Which strand is the hairpin loop formed?
On the NEWLY SYNTHESISED STRAND!
What occurs in Rho dependent termination?
- After RNA polymerase has transcribed RUT sequence (row utilisation sequence)
- Complemenatry sequence of RUT is created on RNA molecule
- This is recognised by the Rho protein
- Then wraps around the protein and is a helicase (also tries to catch up with RNA polymerase -Rho moving along RNA whilst RNA polymerase moving along DNA
- RNA polymerase gets to termination sequence, and pauses…..Rho catches up and unwinds RNA-DNA complex at transcription bubble…RNA, Rho, polymerase ALL FALL OFF
How does Rho unwind the RNA-DNA complex?
By hydrolysing ATP
What is the Rho dependent terminator sequence rich in?
ONLY C’S (not Gs) before the acutal site of termination
How many possible codons are there in the genetic code?
64 possible codons
How many possible reading frames are there and what is a reading frame defined by?
- (in one direction)g frame gives .
- Each reading frame gives different sequency of codons
- only one encodes correct protein
- other ones not likely to encode proteins
- an INITIATION codon –> AUG encodes methionine (MET)
What are the possible termination codons?
UAA ,UAG, UGA
What does the degenerancy in the genetic code mean?
That more than 1 codon can specify an amino acid
Where is the genetic code NOT UNIVERSAL?
In mitochondria (codes for Tryp instead of STOP (UGA( )
How many codons specify amino acids?
61 , because 3 are STOP codons
Where is the code ambiguous?
Where AUG can code for either Met and Start (pretty much unambiguous though)
What is the process of matching amino acids with codons facilitated by?
Carrier molecules (tRNA) (adaptors- bind specific AA and codon) - Ribosomes acting as the strucutral network
Which strucuture and process does Crick;s adaptor hypothesis correspond to?
The transfer RNA with the amino acids and codon (translation)
What did Hiagland and Zamecnik discover in 1956?
Amino acids were activated before incorporated into proteins (attachment of aa to RNA molecule called tRNA to form aminoacyl tRNA)
- tRNA 1st adaptor molecule and tRNA syntherases 2nd adaptor molecules
What actually occurs when amino acids are attached to tRNA?
tRNA synthetase attaches aa (by covalent bond) to tRNA (ATP used)
- Forms covalently bonded aminoacyl tRNA
What are tRNAs?
- small chemical modified RNAs (75-90 nucleotides)
- Unusual bases from chemical modification
- Have a tridimensional shape
- H bonds from same internal base pairing
- Have an adaptor function connecting aas, mRNA and ribosome
`What position do the amino acids attach in the tRNA molecule?
3’ end where the molecule looks like an ‘arm’ stretching out on right hand side
Which enzyme matches amino acid with tRNA?
aminoacyl tRNA synthetases
- usually 20 different enzymes specific to each of the 20 aas
Can many aminoacyl tRNA synthetases recognise their own tRNA molecules using anticodons?
YES! But this is NOT ALWAYS THE CASE
- Seine-6 diff codons (degenerate)
- tRNA also recognised using segments on acceptor end and bases elsewhere in molecule
What happens when some organisms don’t have genes for all the 20 aminoacyl tRNA synthetases?
They still use all 20 aas to construct their proteins,
- Use enzymes to modify existing structures to get the correct form
How many mistakes do tRNA synthetases make?
1 in 10 000
What is the second active site in aminoacyl tRNA synthetases for?
To perform editing reaction (1 in 3000 error now)
What is common to ALL tRNA synthetases?
- Active site catalysing covalent attachment of amino acids to tRNAs
- Binding surface for tRNA anticodon
- Binding site for ATP
What does the anticodon arm contain?
Unusual base called inosine (modified from adenosine)
Whst is the tRNA loaded with amino acid called and what is it catalysed by?
Charged tRNA and catalysed by aminoacyl-tRNA synthetase
What provides energy for making the peptide bond?
The ‘permanent’ covalent bond in the tRNA
What are the steps in amino acid activation?
- AA attaches to the phosphate on adenine (alpha carboxyl attacks alpha phosphate)
- ATP then hydrolysed and amino acyl AMP formed
- AMP group then released an high energy bond is formed
- this leaves the amino acid part to bond to tRNA -catalysed by amino acyl tRNA synthetase
How does amino acyl trRNA synthetase make sure that it is targeting the correct anticodon?
‘grabs’ the anticodon and separates it
Do all synthetases have proofreading activity?
NO!! Some do but not all
What is the overall reaction equation for activation of amino acids?
Amino acid + tRNA+ ATP —> 2Pi
What type of interaction do the codon and anticodon have?
Antiparallel orientation from complementary pairing
How do tRNAs read the mRNA?
By DIRECT interaction
Which base of the anticodon is involved i the wobble base pair rule?
Base pair 1 because it has the weakest bond (NOTE: bp 1 of ANTICODON in lecture diagram appears on the right hand side- not left)
Which orientaton do you have the codon in to show the codon/anticodon relationship?
5’–>3’
What is the degeneracy of the genetic code due to?
Partly due to the nature of the anticodon binding- wobble theory
What is the wobble binding?
- Some codon/anticodon interactions can tolerate a mistmatch at the 3’ base of codon (5’ of anticodon) whcih is called wobble
- doesn’t allow genetic code to be ambiguous (Can read more than one codon specifying same aa)
- DIffernt wobble base pairs can bind to different anticodon bases
What does the wobble allow?
Alanine codons GCA, GCC and GCU to all be recognised by the same tRNA
How many tRNAs are needed for 61 codons?
Only 31 tRNAs
What does elongation factor Ts and Tu do?
Elongation factor Ts charges Tu with GTP so it is ready to deliver a new tRNA to the ribosome
Which units are prokaryotic ribosomes made form?
50s and 30s units
Which units are eukaryotic ribosomes made from?
60s and 40s
Are prokaryotic or eukarytotic ribosomes smaller?
Prokaryotic
How does ribosomal RNA bind to mRNA in prokaryotes?
- Through a ribosome binding site (RBS)
- It is the 16s RNA of ribosome that binds
How does ribosomal RNA bind to the mRNA in eukaryotes and which section of ribosome binds?
- Through mRNA at the 5’ cap end
- 18s RNA of rRNA binds
What does the large subunit rRNA catalyse?
- Peptide bond formation
What are the general steps in translation?
- Binding of mRNA to small ribosomal subunit
- Binding of initiation tRNA at AUG (tRNAiMet in prokarytoes)
- Assembly of the complete initiation complex
What does initiation in prokaryotes involve?
- IF is the initiaion factor
- Prokaryotes carry a special form of the of methianinie (N methianine)
- Shine-balgano sequence binds on mRNA to 16s rRNA
According to leningher, what arethe 3 specific steps of initiation of translation in prokaryotes?
- 30s subunit binds IF1 and IF3 then the mRNA
- IF2-GTP binds the 30 s subunit and recruits fMet-tRNA (fmet)—-> base pairs wirth start codon
- 50s subunit associates, IF2 hydrolyses GTP and IF1, IF2 and IF3 dissociate, leaving 70s initiation complex
What is eIF (initiation of translation) ?
It is the eukaryotic initiation factor and has the same role as IF2; forms complex with initiator tRNAi(met) and complex binds to the P site of the small rRNA subunit
In eukaryotes, what binds both ends of the mRNA in initation of translation?
Complex of eIFs
- eIF4E binds 5’ cap
eIF4g binds poly(A) tail
Why do a complex of eIFs bind to the mRNA?
- To ensure the integrity of the mRNA molecule
In initiation of translation, what occurs after GTP–> GDP?
eIF2 falls off
What occurs in the elongation cycle of translation? (4 steps)
- Binding of amino acyl tRNA (charged tRNA)
- Peptide bond formation
- Translocation of ribosome
- Release of de-acylated tRNA
What is the A site in ribosomes?
Where the peptide bond is formed
What is the A site for in the ribosome subunit?
Receptor site
What is the E site for in the ribosome complex?
Exit site
What switches the ribosome back to the normal state after the peptide bond has been formed?
- EGF-G (elongation factor G) hydrolyses GTP to GDP +Pi
then (EF-tu breaks off)
Where does the aminoacyl tRNA bind the ribosome?
In the A site as a complex with EF-Tu (GTP)
What dos the formation of a bond require?
- Peptidyl transferase activity of the ribosome
- When the peptide bond is formed, aminoacyl bond is released
Is there any other type of RNA where the peptide bond is formed?
NO! Exclusively rRNA
What specifically occurs in translocation (3rd step in elongation)?
- After peptide bond is formed…
- binding of elongation factor is E to the A site is coupled to the conformation change required for translocation to take place
(aminoacyl tRNA moves to the A site and tRNA carrying the growing polypeptide chain moves to the P site
What occurs in the termination of translation?
- Stop codon reached (UAG)
- Protein release factor binds to stop codon (peptidotransferase adds H2O molecule to aminoacyltransferase
- at some time ribosome and tRNA DISSOCIATE
What are the 4 simple steps of termination of translation?
- STOP codon
- Release factor
- Deacylation (release from the last RNA)
- Dissociation of the ribosome
Which error rate is the highest out of transcription, translation and replication?
Transcription and translation hade much higher error rate than replication because of redundancy in code.
How do some antibiotics work?
By blocking events in transltion
How come human ribosomes are unaffected in antibiotics?
Strucutral differences b/w human ribosomes and prokaryotes
BUT some antibiotics harm eukaryotes such as chloramphenicol
What is the action of chloromycetin?
The formation of peptide bonds is inhibited
What is the concept of polyribosomes?
- ribosomes work in a production line
- more than one ribosome moves along a chain at a time
- simultaneous translation of mRNA by multiple ribosomes
Why is eukaryote polysome circular?
- eIFs (bind to the 5’ cap and poly A tail)
- This allows the ribosome machinery to check integrity (if mRNA broken, then won’t have caps and won’t encode)
What is spaciotemporal regualtion?
- In prokaryotes everything will happen simultaneously because there is no nucleus BUT in eukaryotes, there is nucleus so must travel out; spatially separated
Where is the shine del gano sequence present in intiation of translation?
Only in prokaryotes and helps the ribosome to bind (ribososme binding site)
What are some post translational modifications in prokaryotes?
( possible chemical)
- Removal of formyl group on fMet
- aa modifications (less diverse compared to eukaryotes)
What are some post translational modifications in eukaryotes?
Large number of chemical modifications
- proteolytic maturation (propeptide removal)
- complex folding process (chaperone assisted, disulphide bonds)
- aa modifications (phosphorylation)
Where are desitnations for newly translated polypeptides in eukaryotic cells?
- Finish and release to organelles
2. Stall translation and translocate to ER
In eukaryotic cells, where are the polypeptides synthesised?
In the cytoplasm (ribosomes in cytoplasm)
Do all polypeptides fold spontaneously?
Not all, some need to be folded by chaperones (big protein complexes)
What is a postranslation modification that occurs to the polypeptide?
Addition of amino acids such as ubiquitin (one or multiple units)
What is the function of a proteosome?
It is a protein complex that degrades the polypeptides when they are not needed
- (ATP driven)
What happens in alzheimers and parkingsons with proteosomes?
The proteosomes do not recognise that a protein has correct folding when it should’ve…
What are the limitations of microarrays?
- Only shows issues at the mRNA level not the protein level
- You need prior knowledge of sequences of the gene you want to identify (so if you haven’t sequenced a gene you can’t study it)
What is true about a microarray?
It is a slide attached with high density array of immobilized DNA oligomers representing entire genome of species under study
- Each DNA oligomer is spotted on the slide and serves as a probe for binding to a unique complementary DNA (cDNA)
- Most commonly used DNA profiling method
What is a conditional/facultative gene?
- Controlled as transcription is needed
What is a constitutive gene?
- Transcribed continually
What is MicroRNA and Small Interfering RNAs (siRNAs) important for?
- Inhibiting mRNA translation (silencing genes)
- ## Form a RISC (RNA-Induced Silencing Complex)
Where can glycosylatyion and disulfide bond formation occur ?
- In the ER
