HRR: transcription I Flashcards
___ polymerases have a proofreading function, ___ do not
DNA; RNA
What direction in RNA polymerase reading DNA? What direction does it synthesize?
Reads 3’ to 5’, synthesizes 5’ to 3’….always!
What is denovo synthesis?
RNA Polymerase initiates RNA synthesis de novo by joining 2 ribonucleotides together to form the first 3’5’ phosphodiester bond. Unlike DNA synthesis, a primer is not required
RNA is synthesized _’ to _’, making bonds _’ to _’
5 to 3; 3 to 5
Specify the protein subunits of prokaryotic RNA Polymerases. Explain how the
sigma (s) subunit is directed to specific promoters to initiate transcription.
Prokaryotes have one type of RNA polymerases, with various subunits. There is an alpha, beta, beta prime, and omega. The beta subunits bind to the DNA and have a helicase to unwind the DNA and the catalytic site to catalyze bond formation.
The sigma subunit is needed to recognize where the promoter is; it looks for the specific sequence and recognizes it, allowing the polymerase to bind in order to begin transcription. There tends to be two consensus sequences
in RNA polymerase in prokaryotes, which subunits make the core enzyme? what makes it a holoenzyme?
a, b, b’; sigma
Define promoter specificity in transcription of prokaryotes
Only one RNA Polymerase exists in prokaryotes. However, the RNA Polymerase holoenzyme can initiate transcription of all specific genes via the sigma subunit
What is the main enzyme used in transcription of DNA?
RNA polymerase; it is a processive enzyme, meaning it stays on the strand instead of dissociating
Differentiate between a closed and open promoter complex during transcription in prokaryotes
Closed: the polymerase has bound to the DNA, but has not unwound the strands yet.
Open: the polymerase has begun to unwind the DNA, forming the open promoter complex.
describe initiation of transcription in prokaryotes
RNA polymerase looks for the promoter site by sliding along the DNA (it has low affinity for other sequences besides the promoters, giving it the ability to do this). The sigma subunit binds to the promoter sequences. The polymerase then begins to unwind the DNA to form an open promoter complex. It can then form the first phosphodiester bond. The sigma subunit then releases from the polymerase.
describe elongation during transcription in prokaryotes
the core RNA moves the transcription bubble along the chromosome. A type II topoisomerase called gyrase is used to create negative supercoils ahead of the bubble to prevent positive supercoiling. A type I topoisomerase is used to relax negative supercoils behind the bubble.
describe termination in transcription of prokaryotes
-Template directed: a GC rich repeat forms a hairpin, halting the transcription reaction. Following the hairpin is a series of U bases, making for weak binding between the strand and they polymerase. These two factors cause the RNA polymerase to dissociate from the template.
-Rho-directed: rho protein Rho protein binds to specific sequences in newly synthesized RNA. Rho protein has ATP-dependent helicase activity that
dissociates RNA from template strand
what does type I RNA polymerase make
produces 45s pre-ribosomal RNA; found in nucleolus specifically
what does type II RNA polymerase make
Type II: produces pre mRNA, primary miRNA, snRNA, lncRNA
what does type III RNA polymerase make
Type III: produces tRNA, 5s rRNA, some snRNA
mitochondrial: all mitochondrial RNAs
Describe the basic components required for eukaryotic RNA Polymerases to initiate
transcription of a gene.
Core RNA polymerase: mundane work; comprised of multiple protein subunits
General transcription factors: required for basal transcription of all genes
Specific transcription factors:
-Activators/repressors: they activate/repress transcription of a specific gene. They bind directly to cis-acting DNA sequences such as enhancers or silencers. They recruit co-regulators.
-Co-regulators: they don’t bind directly to DNA, they instead bind to activators or repressors.
Differentiate between cis-acting elements and trans-acting factors that regulate transcription of individual genes
Cis acting: DNA sequences in the gene itself that regulate it; they are part of the gene. Examples include the core promoter, enhancers, silencers
Trans acting: they come from genes other than the target gene, but they regulate the target. Examples include specific transcription factors.
Describe the basic components required to form a transcriptionally-active RNA Polymerase I complex and how it functions in the production of ribosomes.
Core RNA: 10-12 subunits; binds to the promoter region
Selectivity factor 1: trans-acting element; 4 general transcription factors that binds to DNA sequence in the core promoter of rRNA gene
Upstream binding factor: a specific transcription factor for RNA polymerase I. it’ll bring in other co-regulators and interact with SL1 to promote assembly of an active polymerase
describe rRNA genes
A human diploid cell contains approximately 400 rRNA genes arranged in tandem repeats along the 5 acrocentric chromosomes [13, 14,15, 21, 22] in the nucleolus. Each gene for rRNA is separated by spacer (non transcribed) DNA
Describe the main types of sequence elements in the core promoter of a gene and their
corresponding functions in initiation of transcription by RNA Polymerase II.
Core promoter: minimal stretch of DNA that is sufficient to direct initiation of transcription. They have several consensus sequence elements.
-Initiator, tata box, motif 10 elements, downstream promoter element, BRE
Describe the main components of a pre-initiation complex (PIC) and how they are assembled on the core promoter of all eukaryotic genes.
Core promoter: multimeric enzyme with a c-terminal tail domain containing serine residues that can be phosphorylated.
General transcription factors: TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs).
Mediator complex: It functions in transducing signals from specific transcription factors bound to cis-acting elements to components of the PIC that assemble on the core promoter
what is the primary method of regulating transcription for mRNA using RNA polymerase II?
regulating the assembly of the primary initiation complex
Describe initiation with RNA polymerase II in eukaryotes
TFIID (basal transcription factor) directs assembly and helps identify the transcription start site
TFIIH: has multiple subunits and 2 main functions:
-Helicase activity: unwinding DNA strands in the core promoter
-Protein kinase activity: phosphorylating the CTD in RNA polymerase II, facilitating promoter release for elongation to occur
TAFs act like a ruler and positions tata box binding proteins (TBP) properly
Describe the role of TFIID
TFIID directs assembly and helps identify the transcription start site
Describe the role of TFIIH
TFIIH: has multiple subunits and 2 main functions:
- Helicase activity: unwinding DNA strands in the core promoter
- Protein kinase activity: phosphorylating the C terminal tail domain (CTD) in RNA polymerase II, facilitating promoter release for elongation to occur
Describe the role of TAFs
TAFs act like a ruler and positions tata box binding proteins (TBP) properly
Describe the role of the C-terminal domain of RNA Polymerase II in the elongation step.
serines on the CTD can be phosphorylated which releases the polymerase core enzyme from the promoter.
Explain how the termination step of transcription is linked to polyadenylation of pre-mRNA
once the strand is transcribed, a polyA tail is added to the 3’ end. this protects the pre-mRNA strand.
