2B: transcriptional and post-transcriptional gene regulation Flashcards
define transcription
the process by which information coded in DNA base sequences is transferred to a complementary RNA strand
what are some differences between DNA replication and RNA transcription? in terms of: ➝ purpose ➝ # of strands read ➝ amount of strand read ➝ type of polymerase ➝ strandedness of resulting molecule ➝ types of bases
➝ purpose: RNA transcript is used in gene expression, DNA replicate used in cell division.
➝ # of strands read: only one of the DNA strands is read in RNA synthesis.
➝ amount of strand read: only a small part in RNA transcription, not the entire strand like in DNA replication.
➝ strandedness: RNA transcription creates single stranded RNA molecule, DNA replication creates double stranded DNA molecule.
➝ type of bases: RNA has U where DNA has T.
what are the three steps of transcription?
① initiation
② elongation
③ termination
define initiation
the first step of transcription where the molecular machinery that carries it out assembles at the promoter and begins synthesizing an RNA copy of the gene
what are the two main parts of protein-coding genes?
➝ promoter
➝ transcription unit
what is the promoter?
a control sequence for transcription (specifies where transcription begins). contains the TATA box. located directly upstream of the transcriptional unit.
what is the transcriptional unit?
the section of the gene that is copied into an RNA molecule. bordered by the transcriptional start and termination sites. located directly downstream of the promoter
what is the TATA box? what does it do?
a sequence of base pairs (TATAAAA), which is ~25 base pairs upstream from the transcription start point. determines where transcription will initiate.
define transcription factors
proteins that recognize and bind to promoters containing a TATA box and then recruit the polymerase
what is the promoter proximal region?
region upstream from the promotor that contains promoter proximal elements
what are promoter proximal elements?
regulatory sequences within the promoter proximal region. proteins bind to these elements and may stimulate or inhibit the rate of transcription initiation
what is the enhancer?
a region some distance away upstream of the promoter proximal region. proteins bind to regulatory sequences within this region and may stimulate or inhibit the rate of transcription initiation
what are general/basal transcription factors?
transcription factors that bind to the promoter in the area of the TATA box and recruit RNA Pol II, creating the transcription initiation complex.
what is the transcription initiation complex? rate of transcription on its own?
RNA Pol II combined with general transcription factors bound to the promoter. its rate of transcription on its own is very low
what are activators?
regulatory proteins that play a role in a positive regulatory system that controls expression of one or more genes.
how do activators that bind to the promoter proximal elements work?
they interact directly with the general transcription factors at the promoter to stimulate initiation, causing more transcripts to be synthesized over a given time
what are motifs?
highly specialized regions within and between domains in proteins that serve specific functions and contribute to the function of the protein.
what is a coactivator/mediator?
a large multiprotein complex that forms a bridge between activators at the enhancer region and the activators at the promoter and promoter proximal region, causing the DNA to form a loop.
what role does the coactivator play in the initiation of transcription?
the interactions between all the elements at the areas the coactivator connects (the enhancer and promoter/proximal regions) stimulate transcription up to its maximal rate. the loop created by the coactivator does not move with transcription.
what are the steps of transcription initiation?
① general transcription factors bind to promoter and recruit RNA Pol II (resulting in low basal level of transcription)
② (optional) transcriptional activator proteins bind to the enhancer regions and the coactivator causes DNA looping (resulting in high level of transcription)
define elongation
the second step of transcription where RNA polymerase moves along the gene, extending the RNA chains
what direction do RNA polymerases synthesize RNA?
5’-3’ (adds new ribonucleotides to 3’-OH)
do RNA polymerases need a primer?
no
what unwinds and rewinds DNA before and after RNA synthesis?
RNA polymerase
what are the 3 RNA polymerases and which type of RNA do they produce?
① RNA polymerase I (RNA Pol I): rRNA
② RNA polymerase II (RNA Pol II): mRNA
③ tRNA polymerase III (RNA Pol III): tRNA
how does transcriptional elongation occur?
➝ RNA Pol moves along the template DNA (reads the template DNA in the 3’-5’ direction)
➝ DNA is unwound in front of the moving RNA Pol and reannealed behind the transcription bubble (all done by RNA Pol)
➝ ribonucleotides are added to the 3’-OH end of the RNA transcript
➝ growing RNA transcript is displaced (detached) from the DNA template strand
define termination
the third/final step of transcription where transcription ends and the RNA molecule (the transcript) and RNA Pol are released from the DNA template
what are the 3 types of termination?
① Rho-independent termination (occurs in prokaryotes)
② Rho-dependent termination (occurs in prokaryotes)
③ cleavage and polyadenylation specific factor (CPSF) (occurs in eukaryotes)
what are the steps of Rho-independent termination?
① the rho-independent terminator sequence contains an inverted repeat, followed by a string of ~6 adenine nucleotides
② the inverted repeats are transcribed into RNA
③ inverted repeats in RNA fold into a G-C hairpin loop, which causes RNA Pol to pause
④ the hydrogen bonds in the A-U base pairs in the ~6 nucleotide A sequence break
⑤ the RNA transcript separate from the template, terminating transcription
how does Rho-dependent termination work?
terminator sequence in mRNA is recognized and bound by the Rho helicase, which unwinds the RNA from the template DNA and RNA polymerase
how does CPSF work?
Cleavage and Polyadenylation Specific Factor: poly-A sequence (the polyadenylation signal) in DNA signals the CPSF to cleave the completed mRNA transcript, thereby separating it from RNA polymerase (the poly-A sequence is NOT transcribed). poly(A) polymerase adds a chain of 50-250 A nucleotides one at a time to the newly created 3’ end (the poly(A) tail)
how do eukaryotes and bacteria compare in transcription of protein-coding genes in terms of gene organization?
gene organization is same, but specific sequences in promoter differ
how do eukaryotes and bacteria compare in transcription of protein-coding genes in terms of initiation?
eukaryotes require transcriptional factors to connect RNA Pol II to the promoter. bacterial RNA polymerase binds directly to DNA
how do eukaryotes and bacteria compare in transcription of protein-coding genes in terms of elongation?
elongation is identical in eukaryotes and bacteria
how do eukaryotes and bacteria compare in transcription of protein-coding genes in terms of termination?
prokaryotic cells use terminators, DNA sequences that stop transcription after they are transcribed. eukaryotic cells stop transcription through CPSF
can more than one process of transcription occur along the length of one gene at once?
yes
how are genes that code for mRNA, tRNA, and rRNA different in eukaryotes?
they are transcribed by different polymerases and have different promoters so the right RNA polymerase and other transcriptional machinery can connect to the area
how are genes that code for mRNA, tRNA, and rRNA different in prokaryotes?
they are all transcribed by the same RNA polymerase and their promoters are essentially the same
what are untranslated ends/regions? their purpose?
the 5’ UTR and 3’UTR are present on the ends of RNA in both eukaryotes and prokaryotes. regulate mRNA stability and translational efficiency.
what is precursor mRNA?
pre-mRNA is the RNA directly after transcription, before it has been transformed into mature mRNA
what is the 5’ cap?
a guanine-containing nucleotide that is reversed so that its 3’-OH group faces the beginning of the molecule rather than the end
when/how is the 5’ cap added to RNA?
it is added to the beginning of pre-mRNA by a capping enzyme soon after RNA Pol II begins transcription
what is the purpose of the 5’ cap?
it protects the mRNA from degradation and is the site where ribosomes attach at the start of translation
what is the poly(A) tail?
a chain of 50-250 A nucleotide on the end of the RNA strand, attached following termination of transcription by poly(A) polymerase
what is the purpose of the poly(A) tail?
enables the mRNA produced from the pre-mRNA to be translated efficiently and protects it from attack by RNA-digesting enzymes in the cytoplasm. protects the mRNA from degradation–the longer the tail the more stable the mRNA
what are introns? in what type of mRNA are they present?
non-protein-coding intervening sequence in RNA that interrupts the protein-coding sequence. present in pre-mRNA but are removed from the mRNA before it becomes mature mRNA
what are exons?
the coding sequences in RNA that are retained in finished mRNA and are expressed.
what is mRNA splicing?
a process that occurs in the nucleus, bu which introns are removed from pre-mRNAs and exons are joined together.
what is the spliceosome?
a complex formed between the pre-mRNA and some small ribonucleoprotein particles
what are snRNPs?
complex of RNA and proteins. located in the nucleus. each consists of a relatively short snRNA (small nuclear RNA) bound to a number of proteins
what are the steps of mRNA splicing?
① snRNAs within snRNP recognize RNA sequences at the intron-exon junctions.
② other snRNPs join to produce a larger complex that loops out the intron and brings the two exon ends close together, forming the active spliceosome
③ the spliceosome cleaves the pre-mRNA at the junction between the 3’ end of the first exon and the 5’ end of the intron. intron is looped back to bond with itself near its 3’ end
④ the spliceosome cleaves the pre-mRNA at the junction between the 5’ end of the second exon and the 3’ end of the intron, releasing the intron and joining the two exons together.
⑤ the released intron loop is degraded by ezymes and the snRNPs move on to splice other mRNAs
what is alternative splicing?
the mechanism of splicing that increases the number and variety of proteins encoded in the DNA without increasing the size of the genome. more than one type of protein can be produced from one stretch of gene containing introns and exons, because some exons may be removed together with the introns depending on the goal protein. because of alternative splicing, exons from one gene may combine in different ways and therefore produce different proteins with different functions.
what is the function of 5’-UTR?
it contains a ribosome binding site (RBS) and Shine Dalgarno sequence (in prokaryotes) or Kozak box (in eukaryotes) that function in translational initiation
what is the ORF?
the open reading frame. the region of mRNA that is translated. bordered on both side by the start codon (which is by the 5’-UTR) and the stop codon (which is by the 3’-UTR)
how is gene expression regulated during pre-mRNA processing?
through alternative splicing regulated by regulatory proteins specific to cell type
how does post-transcriptional gene expression regulation through masking work?
masking proteins bind to mRNA and prevent it from being translated. in order to reactivate the mRNA, other proteins remove the masking proteins
what is one way to change the rate of mRNA breakdown? (thus affecting gene sequence)
changing the 5’ UTR. if a 5’ UTR is swapped from one mRNA to another with a different 5’ UTR, it will have the exact same half life as the original mRNA from which the 5’ UTR was taken.
what is RNAi?
RNA interference. when RNA binds to mRNA and inhibits gene expression through blocking the ribosome or destroying the mRNA
what are miRNAs?
microRNA.
what is the miRNA-induced silencing complex?
miRISC. protein complex containing miRNA that binds to sequences in the 3’ UTRs of target mRNAs
how does the miRISC work?
it binds to sequences in the 3’ UTRs of target mRNAs. if the miRNA and mRNA pair imperfectly, the segment that the miRNA binds to (which is therefore double stranded now, with the miRNA there) blocks ribosomes from translating the mRNA. if the miRNA and mRNA pair perfectly, an enzyme in the protein complex cleaves the mRNA, destroying it
what is the siRISC?
siRNA-Induced Silencing Complex. protein complex containing siRNA. functions the exact same way as the miRISC
what is siRNA?
RNA produced from double stranded RNA that is not encoded by nuclear genes (ex: may be made from RNA from viruses to defend against the virus)
