Unit 7: Prokaryotic Transcription Flashcards
TRUE OR FALSE: DNA is the genetic material of most organisms other than some viruses or viroids that have RNA
TRUE
How do viruses that RNA as genetic material complete transcription?
their genetic material must first be transcribed to DNA first before it can be transcribed
TRUE OR FALSE: RNA is translated into protein and this is unidirectional
TRUE: central dogma theory
What does it mean that translation and transcription is coupled in prokaryotes?
This means that as the mRNA is being transcribed, ribosomes can bind to the newly synthesized messenger RNA and start translation of the mRNA into protein even before the Messenger RNA has been fully transcribed.
Why does coupled translation and transcription not occur in eukaryotes?
In prokaryotes, there is not distinct nucleus hence they can have coupled transcription and translation. In eukaryotes transcription occurs in the nucleus and the mRNA needs to come out into the cytoplasm where the ribosomes can bind and initiate translation.
What is meant by a polyribosome or polisome?
This happens when several ribosomes bind simultaneously to a single emerging RNA molecule and translate it into a protein.
Transcription occurs in what direction? It reads off a template strand that runs in what direction?
Transcription is always in the 5’-3’ direction (non-template strand) and the template it is reading off should be the 3’-5’ strand called the template strand.
The non-template strand is also called what? Why?
the coding strand b/c it will have the same sequence as the mRNA and caries the code for protein sequence. It runs in the 5’-3’
What is an RNA polymerase? What is the function?
an enzyme that synthesizes the RNA using a DNA template (formally described as DNA-dependent RNA polymerase).
Function: to copy one strand of duplex DNA into RNA.
The RNA sequence is complementary to which DNA strand? and identical to which DNA strand?
complementary to template strand
identical to coding strand
Promoter
a region of DNA where RNA polymerase binds to initiate transcription.
Start Point
the position on DNA corresponding to the first base incorporated into RNA
Terminator
a sequence of DNA that causes RNA polymerase to terminate transcription
Transcription Unit
A sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator. it may include more than one gene or multiple genes
When is transcription initiated?
when the RNA polymerase binds to the promoter region.
Sequences prior to the start point of transcription is described as what? Where does numbering of the start point begin?
upstream of transcription (sequences that are in the opposite direction from expression)
numbering begins with the start point -1 and increases as your go further upstream
Sequences after the start point (w/in the transcribed sequence) are described as what? Where does numbering of start point begin?
Downstream - sequences proceeded farther in the direction of expression
numbering begins at +1
What is a primary transcript?
the initial transcript that comes off the ribosome (original unmodified RNA produce corresponding to a transcription unit)
What is the maturation process?
a process in which sequences at the ends of primary transcripts are cleaved off (processed) by endonucleases
-Ribosomal RNA and transfer RNA in both prokaryotes and eukaryotes undergo this process which render them stable.
TRUE OR FALSE: Transcription occurs by base pairing
TRUE: just like replication and translation, transcription also occurs by base pairing in a “bubble” of unpaired DNA
How is a transcription bubble made and In the transcription bubble, which stand is used to direct synthesis of RNA?
RNA polymerase binds to promoter and separates the two strands of DNA to make the transcription bubble.
A template strand is used to direct synthesis of the complementary sequence of RNA
The transcription bubble is about 12-14 basepairs, how long is the RNA-DNA hybrid w/in the bubble?
8 to 9 base pairs
What are the three stages of the transcription reaction?
1) Initiation - RNA poly binds to promoter site on the DNA as a closed complex (closed means DNA stays double stranded). The RNA poly then opens up the DNA helix, in the promoter region, which also includes the start site to form the transcription bubble (which is an open complex and begins synthesis of complementary RNA strand)
2) Elongation - polymerase synthesizes RNA: Involves the RNA strand by the addition of new nucleotides to the 3’ end of the growing chain. As a result there is a RNA-DNA hybrid inside the bubble. Behind the bubble the DNA reforms the double strand helix.
3) Termination - when transcription stops and RNA poly and RNA are released. The polymerase recognizes certain sequences called terminator sequences that signal transcription to stop further addition of nucleotides to the RNA chain. No more phosphodiester bonds formed and the bubble collapses.
Why is the initiation stage sometimes extended for a short period of time before the elongation stage?
because there may be several abortive attempts where short RNA transcripts of about 10 nucleotides are synthesized as the polymerase stays bound to the promoter. The short nucleotides are released and synthesis is restarted until the polymerase can finally extend the chain and clear the promoter.
TRUE OR FALSE: In bacterial there are multiple RNA polymerase that transcribe messenger RNA, ribosomal RNA, and transfer RNA
FALSE: In bacteria there is a single RNA polymerase that transcribes Messenger RNA, ribosomal RNA and transfer RNA. unlike eukaryotes that have three different polymerases; Pol I, Pol II, and Pol III.
What is a holoenzyme?
the RNA polymerase form that is competent to initiate transcription. It consists of five subunits of the core enzyme and a sigma factor.
The core enzyme is made up of two alpha subunits, beta, beta-prime, and omega subunits
What is the C-terminal domain (CTD)?
the domain of RNA polymerase that is involved in stimulating transcription by contact w/regulatory proteins.
-the two alpha subunits of the holoenzyme recognize the promoter via the c-terminal domains.
The core enzyme is made up of five subunits, what are the roles of these subunits?
1 and 2) Two alpha subunits - form a dimer and serve as a scaffold for the assembly of the core enzyme. Also recognize the promoter via its c-terminal domain and serve as binding sites for many factors that regulate transcription.
3 and 4) beta and beta prime - form the bulk of the core enzyme and is the catalytic center. mutations in the genes encoding for these two subunits affect all stages of transcriptions. BB’ form a crablike claw in which the DNA lies
5) Omega subunit - involved ini enzyme assembly and regulatory function
What is the role of the sigma factor?
the sigma factor is primarily responsible for promoter recognition and ensures that the RNA poly initiates transcription from specific sites but also reduces binding of RNA poly to non-specific sites.
-also important in the melting reaction of DNA
The core enzyme of the RNA polymerase binds generally to DNA through what kind of association?
through electrostatic interactions as protein is basic and binds to DNA which is acidic.
TRUE OR FALSE: The core enzyme can recognize promoters
FALSE: the core enzyme cannot recognize promoter an needs sigma factor, which recognizes promoters
Why does the sigma factor change the DNA binding properties of RNA polymerase?
so that its affinity for general DNA is reduced and its affinity for promoter is increased.
-The dissociation constant for the core enzyme to DNA is reduced by a factor of 10,000 and at the same time the Association constant of the holoenzyme to the promoter is increased thousand fold. it also increases the half life of the complex, thus contributing to its stability as well
When is the sigma factor usually released from the core enzyme?
When the RNA chain reaches less than ~10 nucleotides in length, leaving the core enzyme responsible for elongation
How does a polymerase find promoter sequences among the four million base pairs in the e.coli genome?
Three proposed mechanisms:
1) the enzyme may move in a one-dimensional random walk along the DNA (SLIDING)
2) given the intricately folded nature of the chromosome in bacterial nucleoid, having bound to one sequence on the chromosome the enzyme is now closer to other sites, reducing the time needed for dissociation and rebinding to another site (intersegment transfer or hopping)
3) while bound nonspecifically to one site, the enzyme may exchange DNA sites until a promoter is found (direct transfer/intrasegment transfer )
Initially, the initiation complex w/the sigma factor cover how many base pairs? What happens when sigma dissociates
Initially: -75 base pairs (region -55 to +20)
when sigma dissociates: the core enzyme contracts to -30 and as the enzyme moves forward a few base pairs, the core enzyme compacts further forming the elongation complex
Initial elongation complex forms at ____ bases, may lose sigma, and loses contacts from ____ to _____.
forms at 10 bases
loses contact from -35 to -55
General elongation complex forms at ____ to _____ bases and covers ____ to _____ base pairs.
forms at 15 to 20 bases and covers 30-40 base pairs
What is a ternary complex?
the complex in initiation of transcription that consists of RNA poly , DNA, sigma factor, and core enzyme. as well as dinucleotide that represents the first to bases in the RNA product.
What is meant by abortive initiations? When does this occur in transcription?
After the DNA template is positioned properly in the enzyme complex, the next step is the incorporation of the first two nucleotides and formation of the phosphodiester bond to form the ternary complex.
-After each base is added, there is a certain probability that the enzyme will release the RNA chain resulting in abortive initiation products
After release of the abortive product, the enzyme begins synthesizing RNA at position +1. This is repeated until the polymerase succeeds in escaping the promoter
What is a conserved sequence?
sequences in which particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations
From the slides: if one was to compare sequences from different examples of the protein or a particular nucleic acid and you would find that the same individual bases or amino acids are always found at the same location, one would call this a conserved sequence.
TRUE OR FALSE: A promoter is defined by the presence of short conserved sequences at specific locations
FALSE:
A promoter is defined by the presence of short CONSENSUS sequences at specific locations
What are the two six base pair elements that are key to the recognition of the promoter region?
1) the TATAAT sequence that is centered on -10 base pairs upstream of the start site. This is called -10 element, TATA box, or pribnow box
2) the TTGACA sequence which is -35 base pairs upstream the start point. called the -35element.
both of these factors bind to the sigma factor
What is the distance between the -10 element and -35 element ? Why is this an important feature ?
The distance is about 16 to 19 base pairs and is conserved in about 90% of promoters.
The distance is critical b/c given the helical nature of the DNA it determines the appropriate separation of the two interacting regions in RNA polymerase and also the geometrical orientation of the two sites w/respect to one another
What is the UP element?
a sequence in bacteria adjacent to the promoter, 20 base pair upstream of the -35 element, that enhances transcription
-it is believed to interact w/the c-terminal domains of the two alpha subunits and ultimately enhance transcription
What is the discriminant element?
The sequences on either side of the -10 element label extended ten element which is upstream of the TATA box and the other element downstream of the TATA box called the discriminant element also interacts with RNA polymerase and contributes to promoter efficiency.
What are the additional elements that can affect promoter efficiency?
- UP element
- discriminant element
- down mutations
- up mutations
