transcription Flashcards
RNA structures
primary: nucleotide sequence
secondary: folding due to H-bonding between complementary bases on the same strand
tertiary: have intrastrand binding
quaternary: RNAs interact as functional units
- due to all these structures RNA has more functions than RNA
Why is RNA less stable than DNA
- presence of a 2’-OH group in ribose, causes it to react intramolecularly resulting at the 3’OH site resulting in phosphate bond breakage
- single-stranded
Why do we use an unstable RNA rather than DNA?
- a carry on from evolution (RNA evolved first)
- can form many tertiary structures allowing it to have many different functions
- easily temporary and degraded molecule offers a way of controlling its level (ex: shutting off expression)
coding region
tells you which amino acid to put in your proteins
coding region and gene expression in prokaryotes
- often a single continuous unit
- transcription, translation and mRNA degradation occur simultaneously
- DNA is free in the cytoplasm
- ribosomes bind to mRNA while being synthesized and start making protein
what do ribosomes translate
- they translate the RNA as its being synthesized from DNA to save time
- never translate the DNA because you don’t want to damage it
introns and exons
exons: protein-coding segments
introns: non-coding segments
environments of DNA and RNA in eukaryotes
- transcription is in the nucleus
- translation is in the cytoplasm
- mRNA has to be modified before it gets translated
- ## environment is more hostile to mRNA
At what rate does translation happen after transcription in eukaryotes
- not coupled like in prokaryotes!
- RNA transcripts are made in the nucleus then are transported to the cytoplasm
- the first RNA made is a primary transcripts, when introns are removed the mRNA is able to come out of the nucleus
transfer RNAs (tRNA)
- adaptors between amino acids and the codons in mRNA
- involved in translation - translate the genetic code to protein
- clover shaped
messenger RNAs (mRNA)
- intermediates that carry genetic information from DNA to the ribosomes
- usually linear
ribosomal RNA (rRNA)
- structural and catalytic components of ribosomes
- circular, binds to mRNA
types of RNAs only found in eukaryotes
- small nuclear RNAs (snRNA, snoRNA)
- micro RNAs (miRNA, siRNA, Crispr RNA)
- long noncoding RNA
RNA synthesis
- happens in 5’ to 3’ direction using 3’ to 5’ DNA template strand, complementary and anti-parallel to DNA template strand
- if you want RNA to have the same sequence as strand A make it from strand B
coding vs non-coding DNA strand
coding:
- strand you want to copy sequence of
- aka “non-template” or “sense” strand
non-coding:
- strand you use as a template for mRNA (opposite sequence)
- aka “template” or “antisense” strand
why can transcription utilize either DNA strand
- there are multiple genes on a chromosome that are located on either strand
- no matter which strand contains the gene, transcription will always occur in the 5’ to 3’ direction
transcription as a chemical reaction
RNAn + rNTP -> RNAn+1 + PPi
general features of RNA synthesis
- precursors are rNTPs
- only one strand of the DNA is used as the template
- catalyzed by RNA polymerase
- RNA molecule is identical to non-template 5’ to 3’ strand and complimentary to 3’ to 5’ template
structure of gene for transcription in prokaryotes
contains…
- promotor
- transcription start site
- RNA-coding region
- terminator
- transcription termination site
promotor region
- regulates the rate of transcription
- where RNA polymerase binds and initiates transcription from
terminator
- signals transcription to stop
- is encoded in the RNA (unlike promotor)
steps in prokaryotic transcription
- initiation
- elongation
- termination
initiation
- RNA polymerase binds, unwinds and joins the first 2 nucleotides
- initiation of RNA synthesis DOES NOT require a primer
elongation
- complementary nucleotides continue to be added
- localized DNA unwinding ahead of RNA polymerase generates a transcription bubble
- transcription bubble moves with RNA polymerase and unwound DNA behind it rewinds, RNA starts to stick out
termination
- transcription stops when RNA polymerase reaches the “terminator” region of the gene
- newly synthesized RNA together with RNA polymerase is released
components of E. coli RNA polymerase
core (a2,B,B’,w): transcribes any DNA sequence - not gene specific
holoenzyme(a2,B,B’,w,o): specific for transcribing genes
alpha subunit of RNA poly
involved in assembly of the tetrameric core
beta subunit of RNA poly
contains the ribonucleoside triphosphate (rNTP) binding site
beta-prime subunit of RNA poly
contains the DNA template binding region
omega subunit of RNA poly
helps to stabilize the tetrameric core
sigma subunit* of RNA poly
binds to the RNA poly tetrameric core, assists in correct initiation of transcription - specifically at promoter region
- give the RNA poly specify for a gene
initiation of transcription in prokaryotes
- recognition of 2 important promoter sequences
a -35 element: 5’TTGACA3’
a -10 element: TATAAT box - transcription initiates about 5-9 base pairs down from -10 sequence, the +1 position is a purine
transcription elongation in prokaryotes
- occurs when sigma factor is released in order for RNA poly to move along template strand
transcription termination in prokaryotes
- rho-independent (doesn’t use terminator proteins)
- commons mechanism is weak H-bonding at U:A residues that allows mRNA to release from DNA when RNA poly pauses at terminator
- RNA hybridizes with itself
polymerases involved in eukaryotic transcription
RNA poly I: transcribes larger rRNAs
RNA poly II: transcribes pre-mRNA, some snRNAs, snoRNAs, some miRNAs
RNA poly III: transcribes tRNAs, small rRNAs, some snRNAs, some miRNAs
specific promoter sequences for genes transcribed by RNA poly I, II and III
- ## promoter-specific accessory proteins recognize each specific type of promotor and recruit appropriate poly for transcription
activators
- specific for a gene and bind general transcription factors so they can bind DNA. to a particular gene
promotors in eukaryotes
- consists of a regulatory promoter and a core promoter
- core promotor contains -35, -25, +1 and +30 sequences
-35: TFIIB recognition element
-25: TATA box
+1: initiator element
+30: downstream core promoter
initiation of transcription in eukaryotes
- involves assembly of general transcription factors
- TFIID assembles first at the TATA box followed by the remaining TFs
- this forms the preinitiation complex
- the “mediator” permits interactions with other activator proteins bound to regulatory regions or enhancers
- DNA loops out allowing bound proteins to interact with BTA
core promoters in eukaryotes
- assemble the transcriptional machinery, but enhancers determine how efficiently they transcribe
distinct enhancers
- contain different cofactors which can increase or not increase transcription by RNA poly II
- most are far removed from the promoters they influence and must bend the DNA to interact with promoter
elongation in transcription for eukaryotes
- many of the general transcription factors remain at the promoter for quick re-initiation with a new pol.II
- a transcription bubble is generated by RNA:DNA binding
- ensures free RNA3’-OH terminus is available for new rNTP addition
termination of transcription in eukaryotes
- involves cleavage of pre-mRNA and 5’ to 3’ degradation of remaining RNA
- terminates when Rat1 exonuclease reaches RNA Poly
Colinerity
- in prokaryotes, the coding region of a gene is not interrupted: the sequence of the gene is co-linear with the amino acid sequence
- the number of nucleotides in the gene is proportional to the number of amino acids in the protein
RNA molecules and processing in prokaryotes
the sequence of mRNA corresponds to the sequence of the gene from which it was transcribed
RNA molecules and processing in eukaryotes
- genes are often interrupted
- the removal of introns is required to form the mRNA that will be translated into a polypeptide
3 main steps of processing: addition of 7-methyl guanosine cap, polyA tail and removal of introns
the 7’methyl guanosine cap (5’ cap)
- occurs early in the elongation process
- added to pre-mRNA via the unique 5’-5’ phosphate linkage
3’ PolyA tail
- pre-mRNA is cleaved 11-30 nt following 5’AAUAAA3’ sequence and then a long string of about 200 “A” residues is added by PolyA polymerase
the removal of introns
- must happen precisely in order to fuse 3’ of one exon to 5’ of the next
- every intron has 2 conceived sequences required for removal
1) 5’ and 3’ splice sequences containing “GU and AG” respectively
2) intron branch point: a concerted “A” residue - happens by RNA splicing via spliceosomes
spliceosomes mechanism
1) snRNP assembly: U1 binds to 5’ splice site and U2 binds to branch site
2) 5’ splice site is cleaved
3) 3’ splice site is cleaved
4) the exons join together
- U1 and U2 draw ends of intron together to cleave
lariat formation
involves a unique linkage between the 5’ phosphate of the G and the 2’OH of the A
- formation is made after 3’ splice site is cleaved
how can one gene make many different proteins?
- splicing of introns can occur in many different ways to give different proteins
alternative splicing
- either 2 introns are removed to yield one mRNA OR 2 introns and an exon are removed to yield a different mRNA
- more common
Multiple 3’ cleavage sites (splicing)
- cleavage may be at 3’ site1 or at 3’ site2
-mRNA products of different lengths are produced after splicing
RNA editing
changes the information content of genes by…
- changing the structures of individual bases
- modification of mRNA by endogenous guide RNAs
- inserting or deleting nucleotides
guide RNAs (gRNA)
- direct the insertion of uridine bases into the mRNA by repair polymerase
- gRNA serves as a template for addition, deletion or alteration of bases
- ## makes new codons that specify new amino acids in the protein
editing of apoplipoprotein-B mRNA
- RNA editing of ApoB changes C to U converting glutamine codons (CAA) to stop codons (UAA) which truncates the protein and gives it a different function with respect to lipid binding
transfer RNA (tRNA)
- clover-shaped adaptors between the AAs and codons in mRNA
- the anticodon of tRNA pair with the codon of mRNA
- contain modified ribonucleotides
ribosomal RNA (rRNA)
- key components of the ribosome
- synthesis of rRNA and ribosomes happens in the nucleolus
- in prokaryotes there is no nucleolus so this process happens in the cytoplasm
synthesis and processing ribosomal RNAs
- methyl groups are added to specific bases and to the 2’-carbon atom of some ribose sugars
- the RNA is cleaved into several intermediates and then trimmed
- in prokaryotes made in 1 transcript and spliced out, also forms a tRNA
snRNAs
- act in complexes with proteins
- plays in post-transcriptional processing of RNA such as splicing
snoRNAs
in eukaryotes, guide the enzymatic chemical modifications of rRNAs, tRNAs and snRNAs
small micro RNAs
- regulate the control of gene expression in different ways
