transcription and replication is chemically and enzymatically very similar to replication, how is it different?
- the new strand is made from ribonucleotides, not deoxyribonucleotides
- has RNA polymerase that catalyzes RNA synthesis without the need for a primer
- the RNA product does not remain base-paired to the template DNA strand but is displaced only a few nucleotides behind where each new ribonucleotide is added (therefore, multiple RNA polymerases can transcribe the same genes at the same time, each following closely behind another-allows large number of transcripts of a single gene in a short time)
- less accurate than replication (less proof reading mechanisms)
- copies only a region and makes many copies vs the entire genome and only once per cell cycle
describe the structure of RNA Pol
because RNA pol performs essentially the same reaction in all cells from bacteria to humans, the structure of RNA Pol of these organisms share many features
- > mostly all are made up of multiple subunits that perform the same task (some phage, organelle RNA Pols are single subunit)
- > shape of each enzyme resembles a crab claw:
- the two pincers are predominantly the the two largest subunits of each enzyme(B’ and B for bacterial cases and RPB1 and RPB2 for Euk)
- the active site is at the base of these pincers in regions called “active centre cleft” and contains one tightly bound Mg2+ ion
- the active site has many channels to allow rna, dna and ribonucleotides into/out of active centre cleft
compare types of eukaryote RNA Pol and bacteria
bacteria have only a single RNA polymerase :
1. RNA Polymerase core enzyme - capable of synthesizing RNA
eukaryotes have 3:
1. Pol 1 - transcribing specialized, RNA encoding genes (the large rRNA precursor gene)
2. Pol II -transcribes most genes (all protein coding genes)
3. Pol III -transcribing specialized, RNA encoding genes (tRNA genes, some small nuclear RNA genes and the 5S rRNA gene)
* Pol IV and Pol V are found only in plants where they transcribe small interfering RNAs involved in transcriptional splicing (both similar to Pol II)
describe the transcription step of initiation
a promotor= DNA sequence that initially binds the RNA polymerase (with initiation factors) and once promotor-polymerase complex formed it will undergo structural changes required for initiation to proceed
- DNA unwinds where transcription will start producing a transcription bubble of single stranded DNA
- Transcription occurs in 5’->3’ direction and ribonucleotide added to the 3’ end of the growing chain (only one strand acts as the template unlike in replication)
- promotors determine which DNA stretch is transcribed and is the most common step at which regulation is imposed
describe the transcription step of elongation
once RNA Pol has made a short stretch of RNA (~10 bases), it shifts into elongation phase
- RNA Pol further undergoes con. change to tightly bind template more firmly
- the enzyme unwinds the DNA in front and rewinds it behind, it dissociates the growing RNA chain from the template as it moves along AND it performs proofreading functions (All functions from one enzyme)
describe the transcription step of termination
once RNA polymerase has transcribed the length of the gene(s), it must stop transcribing and release the RNA product (as well as dissociating from the DNA itself)
describe the three defined steps of transcription initiation
- initial binding of polymerase to a promotor to form a closed complex (DNA remains double stranded and enzyme bound to one face of helix)
- closed complex undergoes transition to open complex where the DNA strands separates ~14 base pairs around the start site to form bubble exposing template strand and allows first 2 RNA bases to be joined. “initial transcribing complex” adds first 10 bases inefficiently and decision is made to either abort or elongate. the enzyme often releases short transcripts (each less than 10 nucleotides) and then begins synthesis again
- promotor escape-
once an enzyme makes a transcript longer than 10 nucleotides, it is said to have escaped the promotor and has formed a stable ternary complex, containing enzyme, DNA and RNA. this is the transition to the elongation phase
what is the bacterial RNA polymerase holoenzyme ?
it is the form of enzyme transformed from the bacterial core RNA Polymerase that only initiates transcription at a promotor site because an initiation factor (called a sigma protein) was added to it
-the dominant sigma factor in E coli is Sigma^70
describe the structure of a promotor that is recognized by a RNA polymerase enzyme in which sigma^70 is added to it
the promotor has two conserved sequences each 6 nucleotides long and separated by a nonspecific stretch of 17-19 nucleotides
- the two defined sequences are centred at ~10bp and ~35bp upstream the site where RNA synthesis starts
- the two sequences are thus called -10 and -35 regions or elements (these sequences are not identical for all sigma^70 promotors)
what are consensus sequences?
they are sequences derived from comparing many different promotors and reflects preferred -10 and -35 regions, separated by the optimal spacing of 17 bp
(few promotors have this exact sequence but most differ from it by only a few nucleotides)
what is meant by the strength of a promotor and how is its strength influenced
promotors with sequences closer to the consensus are generally “stronger” than those that match less well
- strength= how many transcripts it initiates in a given time
- strength is influenced by how well the promotor binds polymerase initially, how efficiently it supports isomerization and how readily the polymerase escapes the promotor
- this explains why promotors are so heterogeneous: some genes need to be expressed more highly than others
what is an UP-element
it is an additional DNA element that binds RNA polymerase and is found in some strong promotors
-it increases Pol binding by providing an additional specific interaction between the enzyme and the DNA
what is meant by an extended -10 element
Some promotors lack a -35 region and instead have an extended -10 element which comprises of the standard -10 region with an additional short sequence at its upstream end
-extra contacts made between polymerase and this extended region compensate for the missing -35 region
what is a discriminator
found just downstream the -10 element and binds polymerase. the strength of this interaction influences the stability of the complex between the enzyme and promotor
describe the 4 regions of the sigma^70 factor and how they bind to the promotor sequence
sigma 1: recognizes the discriminator by an alpha helix
sigma 2: recognizes -10 element and has helix that has amino acids that interact with bases non template strand to stabilize melted DNA
sigma 3: recognizes the extended -10 element
sigma 4: recognizes -35 element, has two helices that form a DNA binding motif (helix-turn-helix) where one helix inserts into major groove and interacts with the bases in the -35 region and the other lies across the top of the groove contacting the DNA backbone
how is the UP-element of a promotor sequence recognized by a holoenzyme?
unlike the other elements within the promotor, the UP-element is not recognized by sigma but instead recognized by the carboxy-terminal domain of the alpha subunit, called ⍺CTD which is connected to the ⍺NTD (by a flexible linker) which is embedded in the body of the enzyme
describe the melting of the -10 elements by σ region 2
melting is when RNA Pol transitions from closed to open complex and the DNA double strands open to reveal the template and nontemplate strands (melting occurs between positions -11 and +2)
in bacteria, this transition is called isomerization and it is a spontaneous conformational change not requiring ATP
->two bases in the non-template strand of the -10 element flip out and insert into pockets of RNA Pol within the σ protein (these interactions stabilize the single stranded form of the -10 element and derive melting of the promotor region)
-isomerization is irreversible and usually means transcription will initiate (in contrast, closed complex formation is readily reversible)
describe the 5 channels of RNA Polymerase
the rNTP-uptake channel allows ribonucleotides to enter the active centre
the RNA exit channel allows the growing RNA chain to leave the enzyme as it is synthesized during elongation
the remaining three channels allow DNA to enter and exit from the enzyme:
the downstream DNA channel(between the pincers) allows downstream/yet to be transcribed ddDNA to enter the active centre cleft
the non-template strand exits the active centre cleft through the non-template-strand (NT) channel and travels across the surface of the enzyme
the template strand follows path through the active centre cleft and exits through the template-strand (T) channel
*the double helix reforms at -11 in the upstream DNA behind the Pol enzyme
describe the role of the σ subunits 1.1 region during Pol closed and open complex formation
acts as a molecular mimic:
blocks active site in closed conformation and moves out of the way during open complex formation
why is it such an impressive feat that RNA Pol can initiate a new RNA chain on a DNA template without a primer
because it requires DNA template strand be brought into Pol active site and held stably in a helical conformation and the initiating ribonucleotide be brought into the active site and be held stably on the template while the next NTA is presented with correct geometry for the polymerization to occur.
- > this is difficult partly because RNA Pol starts most transcripts with only an A which bind the T on the template strand with only 2 H bonds
- initiating rNTP is held tightly in correct orientation by extensive interaction of the holoenzymes σ subunit
- this RNA Pol initiating process requires very specific interactions
describe the three general models of how RNA Pol active site translocates along the DNA template during initial abortive cycles of transcription where the enzyme makes short transcripts of ~10 bases
-which model is believed to be correct?
- transient excursion:
brief cycles of forward and reverse translocation of RNA Pol. the enzyme leaves the promotor and translocates a short way along template making a short transcript and then aborts and releases transcript and returns to original position on promotor - inchworming:
RNA Pol active site extended over template, making a short transcript before aborting, retracting active
site to body of the enzyme still at the promotor & restarting synthesis of RNA - scrunching:
RNA Pol scrunches/pulls in and unwinds downstream DNA into stationary RNA Pol enzyme complex, causing single-stranded DNA bulges in the active site
*this model is said to be correct based on experiments
describe how promotor escape happens
=All promoter-RNA Pol and RNA Pol core-δ subunit interactions need to be broken for RNA Pol to escape promoter region
once transcript is longer than 10 bases, it can no longer fit in the enzyme and starts to go through the RNA exit channel. however, there is a region of the σ factor (region 3/4 linker) that acts as a molecular mimic, mimicking RNA. this σ region lies in the middle of the RNA exit channel and must be ejected in order for the growing RNA strand to be elongated out of the enzyme (this process can take the enzyme several attempts and hence the abortive transcripts)
-the molecular mimic may be lost once it is displaced
-then the scrunched DNA is released and rewound. the concomitant collapse of the transcription bubble may be what provides the energy required for RNA Pol to break free from the promotor and dislodge the σ factor from the core
how does the elongation polymerase synthesize RNA
only ~9 bp of rNTPs of the growing RNA chain paired to DNA template at any given time; the remainder of the RNA chain is peeled off and directed out of the enzyme through the RNA exit channel
-the size of the DNA bubble remains constant throughout elongation because as 1 bp is separated ahead of the processing enzyme, 1 bp is formed behind it
describe the two proofreading functions of RNA Polymerase
- pyrophosphorolytic editing:
the enzyme uses its active site in a simple back reaction where it removes the incorrect ribonucleotide by reincorporation of pyrophosphate (PPi) and adds the correct rNTP - hydrolytic editing:
the polymerase back-tracts by one or two nucleotides and cleaves off the RNA product, removing the error-containing sequence (this type of editing is stimulated by Gre factors (the elongation factor) which enhance this type of editing but also serve as elongation stimulating factors (makes sure Pol elongates efficiently and overcomes “arrest”)
