Transcription is, ______ and ______, very similar to DNA replication. Both involve enzymes. Both involve enzymes that synthesize a new strand of nucleic acids complementary to a _____ _____ strand.
- chemically and enzymatically
- DNA template strand
Name four differences between transcription and DNA replication
- Transcription involves making new strands from ribonucleotides as opposed DNA replication which uses deoxyribonucleotides (most notable)
- RNA polymerase does not need a primer; it can initiate transcription de novo (although in vivo, initiation is permitted only at certain sequences)
- The RNA product does not remain base-paired to the DNA template strand
- Transcription, although very accurate, is less accurate than DNA replication (1 mistake in 10,000 nucleotides vs 1 in 10 million)
The RNA product does not remain base-paired to the DNA template strand. Meaning?
An enzyme displaces the growing chain only a few nucleotides behind where each ribonucleotide is added
The discplacement of the growing chain a few nucleotides behind where each ribonucleotide is added critical for the ____ to perform its functions. Furthermore, because this release follows so closely behind the site of polymerization, multiple ____ ______ can transcribe the same gene at the same time, each following closely behind the other
As an RNA product dissociates from the DNA template just behind each advancing RNA polymerase, the two DNA strands _______
The difference in precision between transcription and replication reflects the lac of extensive _______ mechanisms for transcription, although ______ forms of ________ exist for RNA synthesis
It makes sense for the cell to worry more about the ________ of the replication than that of transcription. DNA is a molecule in which the genetic material is ______, and DNA replication is the process by which that genetic material is passed on. Any mistake that arises during ______ can easily be catastrophic: it becomes _______ in the genome of the individual and gets passed on to subsequent generations
Transcription, in contrast to replication, produces only _____ _____ and normally several from each transcribed region. Thus, a mistake during transcription will rarely do more harm than render one out of many _____ ______ defective
- transient copies
- transient transcripts
Transciprion only selectively copies certain parts of the _______ and makes anywhere 100 - 1000s of copies of any given section. In contrast, replication must copy the entire ______ and do so ______ every cell division.
RNA polymerases perform essentially the ______ reactions in all cells, from bacteria to humans. It is thus not surprising that the enzymes from these organisms _____ many features. Where specifically?
- in parts of the enzyme directly involved with catalyzing the synthesis of RNA
Bacteria have ______ RNA polymerase, while eukaryotic cells _____. Name them
- three: RNA pol I, RNA pol II, RNA pol III
Pol II is the most ______ of these polymerase enzymes, it is also the polymerase responsible for _______ most genes, particularly all _____ encoding genes.
Pol I and Pol III are each involved in transcribing specialized _____ encoding genes. Specifically, Pol I transcribes the large _____ precursor gene, whereas Pol III transcribes ______ genes, some small nuclear RNA genes and the 5SrRNA gene.
The bacterial RNA polymerase _____ _____ alone is capable of synthesizing RNA and comprises two copies of the ____ subunit and one of each of ___, ____ and ____ subunits
The core enzyme is closely related to the eukaryotic polymerases. Name the counterparts for these core enzymes for either Pol I, Pol II or Pol III
The bacterial and yeast enzymes share an overall _____ and _____. This is more true for the internal parts near the _____ ______ and less so for on the ________. Does this make sense? Why or why not?
- shape and organization
- active site
- It makes sense because the internal parts of the enzyme are involved in synthesis of RNA on a DNA substrate (uniform in all organisms). However, peripheral regions of the enzyme are involved in interactions with other proteins and these differ in euk cells compared with prok cells
Overall, the shape of each enzyme resembles a crab claw, (like the hand of DNA pol). The two _______ of the crab claw are made predominantly of of the two _____ subunits of each enzyme. It is ___ and ____ for prokaryotes and _____ and _____ for eukaryotic enzymes
- β & β'
- RPB1 and RPB2
The active site which is made up of regions from both subunits, is found at he base of the pincers within a region called the ______ _____ ______. The active site works according to the two ____ ____catalytic mechanism for nucleotide addition proposed for all types of polymerases
- active center cleft
- metal ion
To transcribe a gene, RNA polymerase proceeds through a series of well defined steps grouped into three phases. Name them
A ______ is the DNA sequence that initially binds the RNA polymerase (together with any initiation factors required) [**in initiation]
Initiation story (6)
- Once the promoter-polymerase complex is formed, it undergoes structural changes required for initiation
- Like replication initiation, DNA around the point where transcription starts will begin unwinding
- The base pairs are disrupted, producing a transcription bubble of single stranded DNA (like DNA repl. it goes in a 5' to 3' direction)
- Unlike replication, only one of the DNA strands acts as a template on which the RNA strand is built
- Because RNA polymerase binds promoters in a defined orientation, the same strand is always transcribed from a given promoter
- The choice of promoter determines which stretch of DNA is transcribed and is the most common step at which regulation is imposed
When does elongation begin
once the RNA polymerase has synthesized a short stretch of RNA (~10 bases), elongation begins
Elongation story (4)
- During elongation, RNA synthesis occurs
- The enzyme unwinds the DNA in front and reanneals it behind
- It dissociates the growing RNA chain from the template as it moves along
- It performs proofreading functions
Once the polymerase has transcribed the length of the gene, it must stop and ______ the RNA product and _______ from the DNA itself, this is called ________. In some cells, specific ______ trigger this, in others it is less clear.
Transcription initiation can be broken down into ______ steps. The first is called _____ complex, the second is called ______ complex and the third is called ______ ______ complex
- initial transcribing
When an enzyme makes a transcript longer than ____ _______, it is said to have escaped the promoter. At this point, it has formed a stable ternary complex, containing ______, ____ and ____. This is the transition to the ______ phase
- 10 nucleotides
The bacterial core RNA polymerase can initiate transcription at any point on a DNA molecule, and this can be shown in vitro using purified _____ _____. However, in cells, polymerase initiates transcription only at _______.
It is the addition of an initiation factor called ____ that converts core enzymes into the form that initiates only at promoters. This form of the enzyme is called the ____ _____ _______.
- RNA polymerase holoenzyme
Promoters recognized by polymerase containing σ share the following characteristic
two conserved sequences each of 6 nucleotides (separated by a nonspecific stretch of 17-19 nucleotides)
The two conserved sequences of 6 nucleotides are centered respectively at ____ and at _____ upstream of the site where _____ ______ starts. What are the sequences called?
- RNA synthesis starts
- -35 region/element and the -10 region/element
Transcription is initiated by RNA polymerase without the need for a ______
Why does DNA replication always need a primer strand?
DNA polymerase does not synthesize new DNA stands (de novo), it can only extend an existing polynucleotide chain.
The primer is typically a short piece of ____ that binds to the ____ ______ strand to form a short hybrid double strand region. DNA polymerase then adds nucleotides to the ______ of the primer.
RNA polymerae can initiate a new RNA chain on a DNA template and does not need a primer. Name two things necessary for this feat
- The DNA template must be brought into the polymerase active site and held stably in a helical conformation
- The initiating ribonucleotide must be brought into the active site and held stably on the template while the next NTP is presented with correct geometry for the chemistry of polymerization to occur.
Bringing the initiating ribonucleotide to the active site and holding it stably on the template while the next NTP is presented is difficult, why?
RNA polymerase starts most transcripts with an A, and that ribonucleotided binds the template nucleotide T with only two hydrogen bonds not the three witnessed between C & G
The elongating polymerase is a _______ machine that syntesizes and proofreads RNA
DNA passes through the elongating enzyme in a manner similar to its passage through the open complex. Thus, double stranded DNA enters the ____ of the enzyme between the ______.
At the opening of the ______ ______, the strands separate to follow different paths through the elongating enzyme before exiting through their respective ______ and re-forming a ____ _____ behind the elongating polymerase.
- catalytic cleft
- double helix
Ribonucleotides enter the _____ _____ of the elongating polymerase through their defined channel and are added to the growing _____ chain under the guidance of the _____ ______ strand.
- active site
- DNA template
Only 8 or 9 nucleotides of growing RNA chain remain base-paired to the _____ ______ at any given time; the remainder of the RNA chain is ______ ______ and directed out of the enzyme through the RNA ______ _______
- DNA template
- peeled off
- exit channel
During elongation, the elongation enzyme adds _____ nucleotide at a time to grow the RNA transcript. Elongation polymerase acts as a molecular _____, advancing in a single step a distance equivalent to a base pair for every _______ it adds to the growing RNA chain. In addition, the size of the bubble (the length of DNA that is not _____ stranded), is ______ throughout elongation. Explain
- as 1 bp is spearated ahead of the processing enzyme, 1bp is formed bhind it.
While synthesizing the transcript, RNA polymerase performs two proofreading functions on that growing transcript, name them
- pyrophosphorolytic editing
- hydrolytic editng
In pyrophosphorolytic editing, the enzyme uses its ______ _____ in a simple back-reaction, to catalyze the removal of an incorrectly inserted ________, by reincorporation of _____. The enzyme can then incorporate another _______ in its place in the growing RNA chain.
- active site
In pyrophosphorolytic editing the enzyme can remove either ______ or ______ bases but spends longer hovering over _______ than _____ making it more accurate. How many nucleotides does it backtrack by?
- correct or incorrect
- one nucleotide
In hydrolytic editing, the polymerase _______ by one or more nucleotides and ______ the RNA product, removing the ______ containing sequence
Hydrolytic editing is stimulated by _____ _____, which both _______ hydrolytic editing function and serve as elongation stimulation factors. Explain
- Gre factors
- They ensure that polymerase elongates efficiently and help overcome arrest at sequences that are difficult to transcribe.
Another group of proteins the ______ proteins join polymerase in the elongation phase and promotes the process of _______ and _______. These proteins are most similar to _____
- Nus proteins
- elongation and termination
Gre factors are equivalent to?
TFIIS (transcription factors of eukaryotic RNA polymerase II)
Under certain circumstances, an elongating RNA pol can become _______ and cease transcribing. One common cause is a ________ DNA strand.
The consequences of arrest if the gene is being transcribed
Catastrophe! The arrested polymerase maes no products and that same enzyme will cause a roadblock to other polymerases attempting to transcribe the same gene
To deal with arrest, the cell has machinery that removes the ______ and at the same time recruits repair enzymes called ______. The repair process is called _____ _____ repair and the protein that does both repair and removal is called ______
- polymerase (arrested)
- transcription-coupled repair
Transcription is terminated by signals within the _____ ______
When RNA polymerase arrests during _______, it can be knocked off DNA by the action of the translocator ________. This termination is triggered by _______ DNA or by other unanticipated hindrances.
Where is termination a normal and important function
at the ends of genes
At the ends of genes, sequences called ______ trigger the elongating polymerase to dissociate from the DNA and release the _____ _____ it has made.
Two types of terminators in bacteria
Rho-dependent and Rho-indpendent
Rho-dependent terminators require a protein called _____ to induce _______. The second cause Rho-independent causes _______ without the involvement of other factors
- Rho: a ring shaped protein with six identical subunits
- rut sites: rather ill-define RNA elements of rho dependent terminators
Rho dependent terminator story:
- Rho binds to single strand RNA as it exits the polymerase
- The protein (rho) also has an ATPase activity, and once attached to the transcript, Rho uses the energy derived from ATP hydrolysis to induce termination. **The precise mechanism of termination remains to be determined
- Either Rho pulls RNA out of the polymerase, resulting in termination; or Rho induces a conformational change in polymerase, causing the enzyme to terminate
How is rho directed to work on particular RNA transcripts?
First, there is some specificity in the sites it bind. Optimally, these sites consist of stretches of ~40 nucleotieds that do not fold into a secondary structure, they are also rich in C residues
Rho-independent terminators, also called _______ __________ because they need no other factors to work, consist of two sequence elements, name them
- intrinsic terminators
- a short inverted repeat (~20nucleotides) followed by a stretch of about eight A:T base pairs
The elements of intrinsic terminators do not affect the polymerase unitl they have been ______. Meaning?
- they function in the RNA rather than in the DNA
When polymerase transcribes an inverted repeat sequence, the resulting RNA can form a _____ _____ structure aka ______ by base pairing itself. How does this cause termination
- stem loop aka hairpin
- disrupting the elongation complex