47 terms

Prokaryote Transcription

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MSX1
Protein that represses transcription, essential fro craniofacial development
PAX9
Protein that controls transcription (TF), required for development of teeth
RNA vs DNA
1. RNA moves info to next step: long unbranched nucleosides (base+sugar) linked by 3'-5' phosphodiester bonds
2. DNA has 2-deoxy D-ribose sugar and RNA has D-robose: this makes RNA more flexible in double helix and less stable
3. RNA is shorter than DNA: RNA has shorter 1/2 life
4. RNA uses U instead of T: C in DNA can get deaminated to U--> thought DNA evolved not to use U but RNA can use U b/c not genetically passed on
5. RNA usually single-stranded and DNA usually double-stranded: RNA can be double-stranded
6. RNA can for 2' and 3' structures which can be very complex: RNA can base pair, DNA can only form double helix
7. RNA associated with different protein complexes and regulate them: can function with Ez activity
tRNA
-example of RNA 2' (base pairing) and 3' structure (clover leaf)
-b/c of base pairing and base stacking
-clover-leaf
-brings amino acids to ribosome
Types of RNA
1. mRNA: most well known and diverse, carries genetic code (proteins)
2. tRNA: carry amino acids to activated form of ribosome for peptide bond formation
3. snRNA: small nuclear, participate in splicing of RNA exon
4. lncRNA: long non-coding RNA
5. miRNA: microRNA, class of small non-coding RNA that bind to complementary mRNA molecules, inhibit translation or sequester mRNA
6. siRNA: small interfering RNA, class of small RNA molecules that bind to mRNA and facilitate its degradation and/or silencing
7. rRNA: major component of the ribosomes and are needed for protein synthesis
Coding RNA
mRNA
mRNA
-coding RNA
-template for protein synthesis: convey genetic information from DNA to ribosome to specify amino acid sequence of protein products
-least abundant RNA <5%
-relatively short in length: heterogeneous in size
-relatively short 1/2 life
- unique structural characteristics in eukaryotes: 5' cap and poly A tail on 3' end, allow recognitions of different regions of cell
rRNA
-non-coding RNA
-most abundant, 80%
-RNA component of ribosome is essential for protein synthesis
-vary in length
-metabolically stable: required for repeated function
-complexes with proteins --> comp. structural features with protein interaction RNA with proteins and allow for translation--> forms 2 subunits (large and small)
-target of many antibiotics--> in selective manner to target translation in prokaryotes
Non-coding tRNA
-adaptor molecule composed of RNA that serves as the physical link b/w mRNA and the amino acid sequence of proteins
-unique
-binds to ribosome
-has the anticodon: 3 bp region that has opposite of the mRNA--> codon transfer to different amino acids
-3' structure allows for the anitcodon
-modified in different ways: amino acid liked direct to tRNA--> carry amino acids to ribosome
Other non-coding RNAs
-snoRNA
-microRNA
-siRNA
-snRNA
-exRNA
-piRNA
-scaRNA
-long ncRNA
-encoded by majority of the genome
-diverse range of functions- mostly regulating information from DNA to protein
General Considerations- shared between eukaryotes and prokaryotes
1. first step of gene expression, where DNA is copied into RNA by RNA polymerase
2. DNA sequence read by RNA polymerase- produces complementary antiparallel RNA, incorporates U instead of T
3. proceeds in same following general steps (on another step)
General Steps
** 1. initiation 2. elongation 3. termination
1. RNA polymerase (with 1+ TF), binds to DNA to initiate RNA synthesis at promoters
2. RNA polymerase creates transcription bubble- separates 2 strands of DNA helix--> done by breaking H bonds b/w the complementary DNA nucleotides
3. RNA polymerase adds RNA nucleotides that are complementary to the DNA strand --> to form the RNA sugar-phosphate backbone
4. H bonds of RNA-DNA helix break--> freeing the newly synthesized RNA strand
5. if the cell has a nucleus (eukaryotes) the RNA may be further processed and sent to cytoplasm (polyadenylation, capping, and splicing)
Difference b/w Prokaryotic and Eukaryotic Transcription
Prokaryotic:
-transcription in cytoplasm
-coupled directly to translation, little processing of RNA
-single Ez synthesizes all RNA, only one RNA pol
Eukaryotic:
-transcription in nucleus
-RNA is processed: mRNA 5' cap, 3' poly A tail, then to cyto
-RNA pol I, II, III synthesize rRNA (I), mRNA (II), tRNA (III)
Promoters
-synthesis of RNA initiated here by Ez RNA polymerase, only one
-2 promoter consensus sequences exist at -10 and -35 regions upstream of initiation site, sequences are directional and the promoter determines direction of transcription
-the -10 consensus sequence in TATAAT and -35 sequence is TTGACA --> recognized and bound by sigma factor of RNA pol --> once sigma factor interacts with these regions on template DNA, the subunits of the core Ez bind to the site
RNA pol
-made of 5 subunits
-alpha:(2) structural role, interacts with regulatory proteins
-beta: contains catalytic site
-beta': binds DNA template
-sigma: recognizes promoter regions
-constantly scanning DNA
Core Enzyme of RNA pol
-alpha2+beta+beta'
-capable of copying DNA into RNA but doesn't initiate at the correct site in the gene
Haloenzyme of RNA pol
-alpha2+beta+beta'+sigma
-correct promoter recognition
-sigma factor recognizes and sends signal to start transcription
-as sigma factor recognizes the promoter, core Ez activity will unwind DNA--> sigma factor falls off --> complex of 4 subunit that scans and synthesizes RNA
Initiation
-transcription bubble: 12 bp long
-after initial stretch of about 8bp has been synthesized the sigma unit is released
-Core Ez binds more strongly to DNA than sigma factor so it stays on
-form of transcription bubble: DNA, nascent RNA and RNA pol core Ez
Elongation
-5'-->3' synthesis
-RNA pol has editing capability (like DNA pol)- in presence of accessory proteins, can sometimes proofread
-fidelity of transcription is much lower than DNA synthesis
-lower fidelity is tolerated, RNA not transmitted to progeny- synthesized by each generation
Termination
2 ways: pho independent and pho dependent
Rho independent
-more simple of 2 ways of termination
-signal found on DNA template strand, consists of a region of inverted sequences (palindromes)
-5' to 3' forward on one strand machines the 5' to 3' on the complementary strand (palindrome)
-palindromic sequences can form a hairpin
-if palindrome is G-C rich (strong bonds) followed by stretch of U's --> form strong 2' structure that has a stronger bond to itself than to DNA or RNA--> falls off of DNA and creates loop
Rho dependent
-rho is a hexametric protein (rho) that possesses ATPase and helices activity
-rho binds to C-rich sequence on RNA transcript --> rho will slow down--> helices moves to 3' end of RNA transcript to unwind DNA-RNA hybrid --> dissociation of rho and RNA pol from RNA
-rho is thought to bind to end of RNA chain and slide along strand towards open complex bubble- when the factor catches pol--> terminates transcription
RNA processing
-important in mRNA in eukaryotes, doesn't happen in eukaryotes
-tRNA and rRNA require extensive mode in pro and eukaryotes
-in prokaryotes: cleavage or addition
Cleavage
-modification in prokaryotes
-nucleases cut RNA
-RNA cleaved into smaller fragments that can have unique functions
-ex. processing of rRN, transcribed as long transcript that gets cleaved into smaller sections
-can happen through loop structure formed in RNA that has sequence similarity
Addition
-modification in prokaryotes
-add nucleotides to formed RNA
-ex. add CCA sequence at end of formed tRNA--> required in all tRNA, critical to link tRNA to amino acids, active process critical for tRNA function
Rifampicin (rifamycin)
-inhibits prokaryotic transcription
-prevents transcription initiation not elongation: binds to beta subunit of RNA pol and prevents synthesis of the first phosphodiester bond
-selectively blocks bacterial transcription: there are 3 nuclear eukaryotic RNA pol with different structures that aren't inhibited
-used to treat tuberculosis: effectively inhibits transcription in Mycobacterium tuberculosis, resistant to many other antibiotics
Operons General
-combo of different genes transcribed together
-functioning unit of genomic DNA containing a cluster of genes under control of single promoter
-includes: 1. promoter (control sites) 2. set of adjacent structural genes 3/ adjacent regulatory DNA element that affect transcription of the structural genes
-enable organisms to regulate the expression of various genes depending on environmental conditions
Polycistronic mRNA
Genes transcribed together into an mRNA strand and either translated together in the cytoplasm
Monocistronic mRNA
Eukaryotic mRNA usually undergoes trans-splicing to create single mRNA's that are translated separately, ex. several strands of mRNA that each encode a single gene product
Regulatory elements in operons
Creates a protein that can come back and regulate promoters or genes directly
Negative Control Operons
Involves binding of a repressor to an adjacent DNA element (operator) to prevent transcription
Positive Control Operons
An activator protein stimulates transcription, usually binding to DNA at a site other than the operator
Attenuation Control
Allow or prevents complete transcription
Lac Operon
-negative inducible and positive control
-inhibition of a repressor promotes transcription
-activator protein promotes transcription
Trp Operon
-negative repressible and attenuated
-activation of a repressor inhibits transcription
-leader peptide that regulates the amount and length of transcript
Lac Operon General
-in E.coli, first to be discovered
-glucose is preferred carbon source for most bacteria, lac operas allows for effective digestion of lactose when glucose isn't available
-when lactose is required, 3 genes are expressed and translated: 1. lacZ (beta-galactosidase), 2. lacY (lactose permease) 3. lacA (galactoside-O-acetyltransferase)
Lac Operon- Negative Control
-in absence of lactose, lacI (lac repressor) stops production of Ez encoded by lac operon
- lacI gene coding for the repressor is nearby the lac operon and is always expressed
-repressor (lacI) binds to operator and interferes with binding of RNA pol to promoter to prevent initiation of transcription
Lac Operon- Presence of Lactose
-Lactose metabolite called allolactose (made by B-galactosidase LacZ) protein
-binds to LacI repressor--> causes structural change that inhibits activity= negative inducible
-RNA pol can then transcribe high levels of LacZ/Y/A
-allolactose is a bi-product of lactose--> feed forward: more lactose, more allolactose, more lactose etc.
Lac Operon- Absence of Glucose
-cAMP is a signal molecule whose prevalence is inversely proportional to that of glucose
-cAMP binds to catabolite activator protein (CAP)- which assists in RNA pol binding to promoter--> assists in Lac gene expression
Lac Operon- Presence of Glucose
-cAMP doesn't bind to CAP--> low transcription
Lac Operon- Negative Inducible
-regulatory repressor protein (lacI) normally bound to operator--> prevents transcription of the genes on the operon
-inducer molecule (allolactose) is present it binds to repressor and changes its conformation so unable to bind to operator
Lac Operon- Positive Control
-Activator protein promotes transcription (CAP) which is activated by cAMP--> induces Lac gene expression when needed
Trp Operon- General
-when Trp is present genes for Trp aren't expressed
-5 structural genes: trpE, trpD, trpC, trpB and trpA--> encode for Trp synthase --> synthesizes Trp
-repressor regulator: trpR
-Trp binds and activates repressor protein encoded by trpR--> blocks transcription of structural genes (negative repressible)
-contains a leader peptide that regulations amount of transcript formed, not like Lac (attenuator control)
Trp Operon- Attenuator Control, 2nd level of control General
-translation coupled to translation and ability for RNA to form stem loops
-transcript for TrpL has 4 short sequences: each complementary to next
-3 hairpins can form: 1-2, 2-3, or 3-4
-the 3-4 hairpin functions as a transcription termination sequence: G-C rich followed by U
-the 2-3 hairpin prevents formation of 3-4 termination sequence
-the 1-2 inhibits formation of 2-3 which allows 3-4 structure
Trp Operon- Attenuator Control Mechanism
-when Trp are high, ribosome translates quickly--> prevents formation of 1-2 and 2-3 hairpins--> allow 3-4 to form--> termination
-part of initial transcript codes for a short polypeptide of 14 amino acids= leader peptide: 2 adjacent Trp residues
-if ribosome tries to translate peptide while Trp levels are low--> stall at either of the 2 trp codons
-stalling leads to formation of 2-3--> prevents termination --> Trp gens activated by modulating secondary structure of RNA
Trp Operon- Negative Repressible
-transcription of TRP operon is normally taking place
-repressor proteins produced by regulator genes: but unable to bind to the operator in normal confirmation
-when Trp levels are high, activation of a repressor inhibits transcription
Trp Operon- Attenuation
-when Trp leaves are high, translation proceeds quickly allowing for distant secondary structures --> creates termination sequence