18 Flashcards
two ways to end transcription
Rho: protein that hekps with terminantion
Signal to terminante however: present on dna, certain sequence that takes a particular shape that tells rna pol to stop (shape is a hairpin loop
two ways:
1) rho dependent
-binds to sequence on rna (RUT SITE- rho utilization site)
-hexameric protein
-once it binds it starts travelloing up rna
-at end of termination, we make a hairpin loop
steps:
Rut site -> binds to rho factor -> starts migrating up rna to where pol is transcribe
-once rna pol sees hairpin structure it pauses (before its fast)
-rho catches up
-rho factor displaces rna pol and we get the end of transcription
2) Rho independent
two characteristics:
1) GC rich self complementary region: forms stem loop strucutre
2) follow by series of U residues
-imagine if it is GC at end instead of A, if u pull it, it will be hard to dissociate because GC has tight bonds, A doesnt so thats why it is A (U) at the end so that it cafn pull it up to make hairpin structure
-inverted repeats are made immediately after the synthesized polyU region, rnap pauses at hairpin loop
rho dependent steps
Rut site -> binds to rho factor -> starts migrating up rna to where pol is transcribe
-once rna pol sees hairpin structure it pauses (before its fast)
-rho catches up
-rho factor displaces rna pol and we get the end of transcription
Hairpin strucutre
-symmetrical sequecne that when they base pair, makes loop structure
both rho dependent and rho independent have this
Rho ind termination
1) Synthesize rna
2) after synthesized poly U region, the inverted repeats are synthesized
3) inverted repeasts base pair
4) pull on AU basepairing, it cant hold on tight so rna falls off
5) rna is released
6) dna reanneals
7) rnap disscoatiates from dna
Attenuation
REGULATED PREMATURE TERMINATION OF TRANSCRIPTIOON
(opposite of things like the lac operon (repressor) which is controlloing transcriotion iniation, this is controlling transcription termin8ation)
-does not involve rho
-utilizes alot of energy
-happens at the ATTENUATOR SITE (specific site at which attenuation happens)
Trp operon ised to explaoin
Regulation of initiation
-trp operon functions to make enzymes that make tryptophan
it is neg regulation
in absense of tryptophan we have a repressor and when there is no tryp we have a repressor that is not activated, it makes tryp
-when there is tryp, it binds to repressor and makes iactive, allows it to bind to the operon, ending transcription of it
Say we have tryp mutants that have normal repressor, normal operator and normal promoter, but there is a deletion of 50 nucleotides found after promoter operator region but before coding sequence (this is the mutation)
-this is able to have high level of trp synthesized EVEN WHEN REPRESSED… why
-this is because the 50 nucleotide deletion occured in the LEADER SEQUENCE, this made the operon always turned on even if thereis a normal functioning operator, repressor, etc
How???
Leader sequence encodes a short peptide: within peptide there is TWO TRPS coded,
-before coding sequenced is reached, there is a short peptide that is made that has two typs in it
-so when trp is HIGH, only the leader sequence is made and nothign else, transcription is stopped
-when tryp is LOW, it lets u continue transcription into coding region
so what does this mean?
when there is high tryp, it leads to PREMATURE TERMINATION OF IT (only leading strand is made and then it is terminated!)
what is the leader sequwndcw (trp op)
162 nucleotides long
found aftger promoter operator region
-codes for poly
What are the regions of the leader sequence and attenuator
1: synthesis of leader peptide (2 trp codons made) (part of leader)
2: can bind with 1 or 3
(1 and 2 can form stem loop or 2 and 3 can make step loop)
3: can base pair with region 2 or 4
(agai 2 and 3 can form stem loop or 3 and 4) (part of attenuator)
4 (part of attenuator)
after 4 there is a poly u
3,4 is used for rho independent termination of transcription
ie if 2 and 3 make a loop, it wont cause end BC the loop is not beside a poly U region so it does not cause the rnap to become unstable
if 1 and 2 do it, 3 and 4 can at the samw time too and vice vera BUT if 2 and 3, then 1 and 4 CANT only 2 and 3 can)
When trp is low
No attenuation
Ribosome translating right after rna pol (no pause remmeber)…
RIbosome has to stall at region 1 on leader thing (with the two trpcodons) undtil it has available trp charged TRNA bc not enough for tryp for rib to go (it looks for typ in the cell,)
this means that 2 and 3 can basepair and make a loop cuase it is on 1
RNAP continues cuase it is not on 3,4 region
therefore clear that translation dependent on transcription
When trp is high
Attenuation is neede cuz we have lots of trp
Rna pol is transcribing , ribosome moves on strand, comes to sequence 1, but thistime doesnt stall and it continues since there is lots of trp, so 1 is free now to basepair with 2 (this doesnt really havge an effecr though becaseu there is no poly U region that follows this ) and 3and 4 can hairpin too…
since 3 and 4 form the ATTENUATOR loop, polyU follows region 4, this destabilizes rnap in combination with the loop
RNAP therefore will terminate before it reaches the trp structural genes (coding genes)
therefore what are the ways in which trp can be regulated
1) Trp gene repressor (in presence of trp will bind to operator)
2) Attenuator sequence: prematurely terminates transcrip which high levels of tryp are present (attenuated rna consists of short sequence)
look at pic on slide 17
Amino acid biosynthetic operons
-also have this pathway
-presence of what u want is regulates wherther u transcribe genes or not
MRNA PROCESSING
capping and polyadenylation
What is Heterogenous nuclear rna vs primary transcripts
HnRNA:
-collection of transcripts made by rnap 2 (rna pol)
-made in one stretch
-can be made into pre-mrna or snrnas
Primary transcripts
=pre-mrna
-tyranscripts that are going to be processed to give mrna
Inrtrons and exons
introns: nevertranslated and spliced out
extrons: joined together
RNA BINDING PROTEINS
-little different than dna binding proteins because single stranded rna doesnt have double strands so
-bindingof these proteins allow for other steps such as splicing and transport
-flat sheets with helixes
mRNA processing
rna is LINEAR, therefore subject to degredation because cell thinks its viral rna
to stop this we have
1) 5’ capping (CTD tail): blocks exonucleases from coming in
-adding 7 methyl-guanosine (7MG) to the 5’5 phosphates
2) Rna Splicing (CTD) in middle
-segments that dont hybridize are the introns that have been spliced out
Hybridizing mrna back to original template will. be much smaller
-more introns than exons in a gene (4-10x)
3)polyadenylation (CTD tail) : fo9und at tail, prevents degredation too
-this is a signal for enzymes to bind to
-poly A tail has. aperfect consensus (AAUAAA)
How: consensus positions endonuclease to cleaveand poly A polymerase attaches A
-CTD role is to allows positioning of enzymes that help it to polymerize and polyadenylation (allow things to bind and process the transcript0)
Note: When tail isphosphorylated it has lots of activities:
capping facrors can bind
PRE MRNA doesnt have these things
As soon as u start transcribing mRNA, put a cap on the 5’ end, as we syntehsize, we make introns and extrons and they are being spliced as it happens, and then at the end a string of A’s is made
-everytime we translate rna, the ribosome takes off an A, so after certain number of translations , rna is degraded because there is no more As to protect
-rna is a wimpy molecule so this is good to make it have a finite lifetime so that it can be preserved well
All processing happens in nucleus
translation in the cytoplasm
What happens if incompletely processed mrna is translated
could be a faulted protein, changes meaning of the coding region
What makes rna have a short life
poly a tail
each time the rna is translated, it looses one of the A’s until eventually it is degraded because there is no more A tail to protect it
If an mrna is not made properly will it leave the nucleus
no, most lilely will not
What is at the beginning of the 5’ cap
7MG
7 met guanisoine
Wjat is the role of CTD
the ctd tail is neccesary to bring enzymes that are needed for these processings
Origin of introns and exons
Exons are usefl cause u can rearrange them in many ways to make blocks of genes
-like lego set, can make variations of things using the same gene
Which genes have no introns
few
histone genes
interferon gene
Mechanisms for splicing
3 waysfor introns
Grouping is based on how they are spliced
1) Splice themselves without enzyme help (ribozymes), g in intro cuts it and then the free intron goes and gets the other ones
2) Ribozyme again: but this time OH on A goes an attackes. a junction G (with a G usually at the place between end of extron and beginning ofintron), the other intron goes and gets the other one off but in the process, the place where te original one came off goes and binds to to the A making a loop
3)Spliceosome: use lots of enzymes to help achieve, similar to second way in that the A loops with the first open end
5 different proteins for this
2 make COmplex A:
U1: Binds to (junction) G
U2: binds to the A
3 bind at other junction:
U4: inhibits 6
U6:
U5:
U1 and U4 come off after
really just know thatit is the same reaction for the third way but it is proteins that make the framework
zSplicesomes proteins are mixtures of proteins and rna, rnas help to position the proteins, produce same archetecture as others i think kidk
First life strted with
rna organism
