The Central Dogma- DNA, RNA and protein-11 Flashcards
Bacterial transcription
How do the expression levels of genes vary?
Gene expression can be regulated at the level of transcription or translation or both.
-When referring to strands, the gene depends on context.
-Each gene is uniquely regulated.
Which strand does mRNA carry information for?
mRNA carries the sense of information.
Non-template/sense strand is a direct copy of mRNA (with U instead of T).
Template/antisense strand is copied to make the sense strand of mRNA.
How does the sense strand depend on context?
The sense strand for gene A is the antisense strand for gene B.
The sense strand for gene B is the antisense strand for gene A.
Why is uracil not found in DNA?
Cytosine can undergo spontaneous deamination to produce uracil.
C pairs with G. U pairs with A.
DNA replication after deamination would replace a C-G base pair with a U-A base pair, which introduces mutations. In DNA, any U is removed by uracil-DNA glycosylase, generating an abasic site, which is removed and repaired by DNA polymerases.
What are the 3 major classes of bacterial RNA?
mRNA
rRNA
tRNA
How many RNA polymerases synthesise the 3 major classes of bacterial RNA?
1
(In eukaryotes, there is a separate RNA polymerase for each class)
What is the roles of mRNA, rRNA and tRNA?
mRNA- encodes proteins.
rRNA- constituents of ribosomes: role in protein synthesis.
tRNA- adaptors between mRNA and amino acids: role in protein synthesis.
What happens in the bacterial transcription unit?
5’ promoter- attracts and binds RNA polymerase. Expect an operator (on-off)
Transcribed (protein coding) sequence(s): often polycistronic as part of an operon.
3’ terminator: signals the stop point for transcription.
-No nucleus so transcription and translation can occur simultaneously.
-Direction of transcription depends on the sense strand.
What is polycistronic?
mRNA corresponding to multiple genes whose expression is controlled by a single promoter and terminator.
What is an operon?
A functioning unit of DNA containing a cluster of genes under the control of a single promoter.
Describe Bacterial RNA polymerase?
A multi-subunit protein complex.
Composed of alpha, beta, beta’ and omega subunits in the ratio of 2:1:1:1.
Addition of a sigma subunit converts the enzyme to holoenzyme. Total:6 subunits.
Where does RNA polymerase bind with DNA?
At promoter sequences.
The core RNA polymerase binds DNA non-specifically and can slide.
A sigma subunit binds to the core polymerase and directs the polymerase holoenzyme to a gene promoter.
What is DNA footprinting? Why is it used? How?
A technique used to identify promoters.
This is done by binding RNA polymerase holoenzyme to DNA in vitro.
Add nuclease/DNase: the DNA is degraded, except for the stretch that is bound to the polymerase, which is protected.
Only a small amount of DNase is used- the aim is partial degradation to generate a ladder of nicked intermediates.
What does a bacterial promoter look like?
Two regions (-10 and -35) are typically protected by RNA polymerase, because polymerase makes two contacts with the promoter.
They are centred around the middle of the base pair sequence on the electrophoresis.
What are conserved elements in DNA footprinting?
-35 sequence: centred around -35bp from the start of transcription.
-10 sequence: centred around -10bp from the start of transcription.
(+1): transcription start (usually either A or G).
How can you read the results of a gel electrophoresis in DNA footprinting?
Conserved elements are protected by RNA polymerase and not showed on the electrophoresis.
Strong promoter sequences are closer (more similar) to the consensus.
How is directionality provided in promoter sequences?
The asymmetry of the promoter sequence provides directionality.
The -10, -35 and +1 consensus sequences are defined on the sense/non-template/non-transcribed strand.
RNA is built in the 5’ -> 3’ direction: new nucleotides are added on the 3’ end, using the antisense strand as a template.
What are the 3 stages of transcription?
Initiation
Elongation
Termination
Give a summary of the 3 stages of transcription?
Initiation- RNA polymerase holoenzyme binds the promoter, opens the DNA double helix and starts to transcribe.
Elongation- the sigma subunit disengages from the holoenzyme, and the core enzyme continues to make a new RNA.
Termination- RNA polymerase core enzyme dissociates from the DNA and transcription halts.
What happens in initiation?
- The core RNA polymerase binds DNA non-specificially and can slide.
- A sigma subunit binds to the core polymerase and directs the polymerase holoenzyme to a promoter, binding to the -10 and -35 regions.
- The polymerase pulls downstream DNA towards itself, scrunching the DNA (abortive initiation) until success where the 10 region is opened, converting the closed promoter complex to an open promoter complex (doesn’t require energy, unlike the action of DNA helicase), negative supercoiling on the left and positive supercoiling on the right. Topoisomerases are used to relieve the problems of supercoiling.
- 12-15 bp are unwound, from within the -10 region to position +2 or 3: the transcriptional start site is exposed. RNA polymerase now makes an RNA copy from the template strand, using base pairing rules (RNA pol does not require a primer, unlike DNA pol). After about 10 nucleotides of RNA syntheis, the sigma factor is exposed and disengages. The RNA polymerase can now elongate the new RNA.
What happens in elongation?
A transcription bubble/proofreading:
- RNA polymerase is highly processive. The RNA:DNA hybrid is ~8bp long. The unwound DNA bubble is ~17bp long.
- Rate of elongation is slow compared to DNA pol, about 50 nucleotides/s.
- RNA polymerase moves left to right.
- Rewinding and unwinding occurs.
- If RNA polymerase mis-incorporates a ribonucleotide it hesitates, then backtracks, removes the nucleotide then continues. Around one mistake occurs every 10^4-10^5 nucleotides, even with such proof-reading functions.
What happens in termination?
Transport in bacteria:
1.Rho (p)-independent: a terminator sequence in the RNA is recognised.
2. p-dependent: requires a p protein to break the RNA:DNA duplex in the transcription bubble.
In both cases, the functioning signals are recognised not in the DNA template, but in the newly synthesis RNA.
What happens in the Rho (p)-independent termination?
A terminator sequence in the RNA is recognised.
DNA encodes STOP signals for transcription.
The simplest is a palindromic GC rich sequence followed by a T rich sequence: transcription terminates either in this run of Ts or just after them.
The RNA transcript can form a base-paired stem (stable, strong G:C bp) with a loop, leaving a tail of 4 or more Us only to participate in the RNA:DNA hybrid in the transcription bubble.
RNA polymerase pauses at the palindrome, the hairpin forms in the transcript and the DNA dissociates because all that is holding the hybrid is a few weak A:U bp.
What happens in the p-dependent termination?
Requires p protein to break the RNA:DNA duplex in the transcription bubble.
P protein is a hexameric helicase that binds to a C-rich G-poor sequence in the RNA, and it uses its helicase activity to chase RNA polymerase. When it catches- it disrupts the DNA:RNA hybrid helix, releasing the RNA.
What is rifampicin?
An inhibitor of prokaryotic transcription.
How does RNA polymerase change the shape of the DNA duplex?
RNA polymerase bends the DNA duplex.
Bending allows the DNA duplex to be opened more easily, this allows the helix to be more open (like a slinky).
What does rifampicin do?
Inhibits RNA polymerase by binding tightly to the RNA exit channel. Therefore it affects initiation (but does not affect RNA polymerase that is already in the elongation stage).
What is a p protein?
Regulates gene expression and binds to a common DNA/RNA site.
What is a holoenzyme?
Formed by the combination of an enzyme and a coenzyme.
Why is such a high error rate tolerated in transcription?
Because RNA errors mean that some transcripts may be mutated, but the majority are not. DNA errors are transmitted to progeny cells- they are stringently repaired.
If the transcript encodes a protein, then most of that protein will be fine, but a small subpopulation may be mutant- and can properly be tolerated.
How many sigma subunits does E. coli have?
Has multiple subunits.
Different sigma factors recognise different promoters, so they provide specificity.
