Unit 10: RNA synthesis and Processing Flashcards
What are the major types of RNA that exist in both prokaryotic and eukaryotic cells?
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Messenger RNA (mRNA)
Describe rRNA
- Ribosomal RNA self assemble with basic proteins to form ribosomes
- non coding RNA
what is the relative abundance of rRNA?
rRNA makes up about 80% of total RNA
Describe tRNA
- tRNA matches genetic information to an amino acid sequence
- tRNA has an acceptor arm, which receives the amino acid and a three-base anticodon, which can form base pairs with the complementary codon in mRNA
Describe mRNA?
mRNA gives the blueprint for amino acid sequence. therefore codes for proteins
what is the relative abundance of tRNA?
tRNA makes up about 15% of all RNA
what is the relative abundance of mRNA
mRNA makes up about 5% of all RNA
what are additional RNA types unique to eukaryotes and their general function?
small nuclear RNA (snRNA)- splicing
micro RNA (miRNA)- regulatory small RNAs, inhibit gene expression
component of signal recognition complex (SRC)- helps with targeting proteins to ER
component of telomerase- maintenance of chromosome ends during replication
Long non-coding RNA (lncRNA)- various regulatory functions
Name the components of a DNA transcription unit
- promoter,
- transcription start site (TSS),
- 5’ UTR (untranslated region)
- 3’ UTR (untranslated region)
- coding sequence,
- terminator
What are the main stages of transcription?
- initiation
- elongation
- termination
How is transcription similar to replication?
- Both need a DNA template
- initiation takes place a specific sites. replication its at the origin. transcription is at the promoter/TSS
- elongation in replication and transcription is the synthesis phase. both synthesize in 5’ to 3’ direction
- termination in replication and transcription happens at specific signals. in replication the replication fork meets or reaches the end of a chromosome
How do transcription and replication differ?
- transcription uses NTPs/rNTPs while replication uses dNTPs
- UTP replaces dTTP in transcription
- transcription limited to segments of the genome
- transcription does not require priming
- only one strand used as template w/in transcription unit
describe the function of a promoter
- its DNA sequences that direct RNA Pol to proper initiation site for transcription
- happens upstream of TSS
- act on the same strand (cis acting)
Is a promoter more complex in eukaryotes or prokaryotes
eukaryotes
Describe strong and weak promoters
Strong promoters bind polymerase more frequently leading to more RNA being synthesized thus more protein production
prokaryotic promoters vs. eukaryotic promoters
prokaryotic:
have a -35 region and a -10 region (called the pribrow box)
have a +1 start site
eukaryotic:
have a TATA box which is at (-31 and -26)
promotes well defined TSS
often paired with initiator element
have CpG island promoters which are clusters of C and G adjacent to one another
- near TSS
- more frequent in constitutively expressed genes (low level expression)
- promote undefined start sites ( dispersed promoters)
- more common than focused promoters in vertebrates
what are the pribrow and TATA box region rich in?
As & Ts
describe the prokaryotic RNA polymerase
- RNA polymerase is made up of a core enzyme and a subunit
- the core enzyme is made up of subunits (pentamer) and a Mg2+ cofactor
- the sigma subunit combined with the core enzyme is the holoenzyme or functional unit
- the polymerase is not functional without the sigma subunit/factor
what does the sigma factor do?
- sigma factor helps recognize the promoter sequence, bind it and initiate melting.
- the polymerase does not need it afterward or once elongation begins and is replaced with elongation factor NusA
- sigma factor binds at the -35 and -10 regions
- sigma70 is the most common
what does RNA pol 1, 2 and 3 do and where are they located?
RNA Pol 1: synthesizes rRNA and is in the nucleolus
RNA Pol 2: synthesizes mRNA, miRNA, and snRNA and is in the nucleoplasm
RNA Pol 3: synthesizes tRNA and some rRNA and is in the nucleoplasm
what are transcription factors and what do they do?
proteins that aid in transcription.
- they position polymerases at initiation sites
- mediate melting
- assemble at initiation site in step-by-step function to form Transcription initiation complex (TIC)
- required for initiation of all polymerases
TFI- helps RNA Pol 1
TFII- helps RNA Pol 2
TFIII- helps RNA Pol 3
How are prokaryotic and eukaryotic RNA polymerases similar to each other and how do they differ from DNA polymerases
similarities:
- DNA-dependent. synthesize RNA based on DNA template
- use rNTPs and Mg2+
- elongate by adding to 3’-OH end of the new RNA. synthesize 5’ to 3’ direction. energy from PPi drives reaction to make it energetically favorable
Different:
- do not require priming b/c of binding to promoter sites initiate synthesis
- 5’ nucleotide does not release PPi. RNA transcripts have a triphosphate at their 5’ end
- very limited proofreading
describe initiation of transcription in prokaryotes (basic steps)
- RNA pol core binds to DNA promoter and closed complex forms at the promoter
- Melts DNA to produce single strand template
- transcription bubble forms
with the open complex, transcription is initiated - abortive initiation
- promoter clearance and sigma factor released
describe the main mechanisms of transcription termination in prokaryotes
- Rho-independent (intrinsic) termination
- no additional protein factor required
- termination is initiated by transcription of endogenous termination sequence
- the secondary structure of RNA dissociates RNAP
- termination sequence in mRNA and a self-complementary region in template followed by a string of As
- A hairpin dsRNA structure formed by RNA transcript disrupts RNA-DNA hybrid and/or interaction with RNA-pol
- mRNA is released
- rehybridization of DNA - Rho dependent termination
- requires mRNA elements and protein factor (p) for termination
- Rho protein has helices function and dissociates transcript from template DNA
- Rho binds to Rut sequence (a specific mRNA seq). it is C-rich in sequence in RNA
- RNA binding activates helices activity
- unwinds hybrid in bubble and separates RNA-DNA hybrid
describe the main differences between transcription in prokaryotes and eukaryotes
- Different gene arrangement
- operons in prokaryotes for coordinated expression
- single genes with intron-exon structures in eukaryotes - several RNAP in eukaryotes transcribing different parts of the genome
- extensive packaging in eukaryotes
- more complex post transcriptional RNA processing in eukaryotes due to the spatial separation of transcription and translation
- spatial separation of transcription and translation in eukaryotes
- complex transcription regulation in eukaryotes
- has more regulatory elements
- more proteins required for initiation and elongation
polycistronic transcript vs. monocistronic
poly= many messages
mono= single message
eukaryotic initiation of transcription compared to prokaryotic transcription
eukaryotic:
Transcription factors help position polymerase at initiation site to form TIC
- helps induce conformational change in DNA
- recruits more transcription factor to complete pre-initiation complex
prokaryotic:
describe termination in eukaryotes
termination signal after transcribing sequence directing polyadenylation
- poly(A) addition site is AAUAAA
- this process is also coupled with 3’-end processing of mRNA
how are RNAs processed in prokaryotes and eukaryotes
Prokaryotes:
1. rRNAs and tRNAs are processed
- synthesized as single transcript with spacer regions (this is 30s pre-RNA)
- pre-RNA is processed by RNAses. the ends are trimmed by exonuclease. and the spacers are removed by endonucleases
- addition of CCA at 3’-end of tRNAs
- methylation of rRNA bases
- extensive base modification in tRNA
- most mRNAs are not processed due to concurrent transcription and translation
eukaryotes:
- all RNAs are processed
- rRNA and tRNA processing are similar to prokaryotes. rRNA synthesis and processing in nucleolus
- mRNA, 5’ and 3’-ends are modified and introns are spliced out
- most primary mRNA gets 5’-end capping, poly(A) at the 3’ end, and splicing.
- some primary transcripts get RNA editing
describe mRNA capping
- the attachment of 7-methylguanylate via 5’-5’ triphosphate linkage
- it is a methylated G
- it is done during RNA synthesis. associated with RNAPII CTD
- it protects 5’-end from enzymatic degradation (phosphates and RNAses)
- facilitates export from the nucleus
- mediates binding to ribosome during translation
describe polyadenylation of mRNAs
- associated with RNAPII CTD
- mediated by poly(A) signal AAUAAA. this tells enzyme to cleave the transcript and polyadenylate the 3’-OH end
- heterogenous length of poly(A) tail
- this increases translation efficiency
- increases mRNA stability by protecting 3’-end from RNAses
- not all mRNAs are polyadenylated (such as histone mRNAs)
What is splicing of eukaryotic mRNA and what does it do?
- splicing is the internal cleavage of transcript to remove the introns and ligate the coding exons
- requires cleaving and reforming phosphodiester bonds
- achieved by two sequential transesterification reactions
- no energy required for bond cleavage
- produces excised lariat intron and spliced exons
what are transesterification reactions
—- fill answer—-
what are the intron-exon boundaries
GU at intron 5’-end and AG at intron 3’-end
what is included in an intron?
GU-5’ and AG-3’
polypyrimidine stretch close to the 3’ site
internal branch site containing A ( about 25-50b from 3’ splice site)
what is the spliceosome? and what does it do?
- protein-RNA complex performing splicing reaction
- snRNAs with conserved secondary structures combine with proteins to make snRNPs (small nuclear ribonucleoprotein particles)
- base pairing with RNA required for reaction
- most non self-splicing introns need spliceosome
what are the consequences of alternative splicing
producing different products
What is the purpose of mRNA editing?
- takes place after synthesis
- includes addition, deletion, and modification of nucleotides
- can alter codons
- ## common in mitochondrial and chloroplast RNAs. rare in higher eukaryotes
