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TYPES OF RNA
- differ in stability and function.
major constituent of the ribosomes which are the site of protein synthesis.
carries the amino acids to the ribosome for assembly into a polypeptide.
serves as the template for the polypeptide, provides the bases that code for the amino acid sequence.
UBIQUITY OF RNAs
- all organisms produce the three basic types of RNA.
- viruses do not produce rRNAs, they take over the host cells ribosomes to make proteins.
- viruses do produce some tRNAs, but also utilize the host tRNAs to a great extent.
- functional ribosomes are made up of two subunits which in turn are made up of rRNA and proteins.
- classified based on size using sedimentation constants (S units).
- differ between prokaryotes and eukaryotes.
PROKARYOTIC ORGANIZATION OF rRNAs
- in prokaryotes produced as one long transcript and then cleaved into the three function pieces.
- there are multiple copies of the RNA genes in the genome.
- lots of intrastrand pairing and methylation of specific bases in the RNA sequence.
EUKARYOTIC ORGANIZATION OF rRNAs
- in eukaryotes the large transcript is coded for in the nucleolar organizer region (NOR).
- repeated up to 200 times in humans.
- in mammals a large 45S transcript which is cleaved into 18S, 5.8S, 28S units.
- serves as the template for the actual amino acid sequence in the resulting protein.
- simple a complementary copy of the coding/templated strand of the cistron.
- generally differs from rRNA and tRNA due to a lower a concentration of G-C and little or no intrastrand pairing.
TYPES OF mRNA
- monocistronic: one cistron; contains the information to code for a single protein.
- polycistronic: multiple cistrons; contains the information to code to several proteins. (limited to prokaryotes).
- new class of RNAs called heterogeneous nuclear RNAs (hnRNA).
- actually a group of large RNA molecules that are the precursor to the functional mRNAs.
- involve a three step maturation process form primary transcript.
- 1. addition of a 7-methylguanosine to the 5' end of the molecule.
- 2. addition of a poly(A) tail to the 3' end (150 to 200 Adenine residues added)
- 3. splicing of the molecule or remove introns and connect exons together.
- maturation is a Co-transcriptional event.
- protiens for capping and splicing are associated with the carboxyl tail domain of the beta subunit of RNA polymerase II.
- the CTD includes multiple repeats of 7 aminoacids and is located near the site where the new RNA molecule emerges.
CAPPING AT 5' END
- addition of 7mG to the 5' end by guanyltransferase through a 5' linkage.
- serves to protect the mRNA from degradation and is also required for proper translation.
- actually three different caps are possible, but all are 7mG and 5'-5'.
POLY (A) TAIL
- up to 200 adenine nucleotides are added at the 3' end via poly (A) polymerase.
- serves to increase the stability and longevity of the mRNA in the cytoplasm.
- the signal for adding a poly A tail is a sequence of AUUAAA or AAUAAA followed by a 20 base skip and endonuclease cutting then adding poly A.
- the pre-mRNA must have introns spliced out. reduces total size by 10 to 20 fold.
- this is a sequence based system using signals in the pre-mRNA.
- 5'GU UACUAAC AG3'
- left branch right
- splicing requires a splicosome which includes a small RNA and a splicozyme.
- model is called Lariat Formation.
- splicing does not follow any specific pattern and thus produces heterogeneous RNAs as different introns are cut out.
- involves 5 different small nuclear RNAs (U) plus over 100 proteins.
- U1 binds at the 5' (GU) sequence and U2 binds at the branch sequence (internal A).
- U4, U5, and U6 complex joins the splicosome bringing the 5' near the internal A of the branch sequence.
- U4, U1, and U6 leaving U5 and U2 to make the first cut and attach the 5' GU to the internal A of the branch sequence.
- after the first splice the second cut occurs at the 3' AG sequence resulting in the formation of the intron "lariat".
- simultaneously the two exons are joined to complete the removal of the intron.
- the lariat with U5 and U2 still associated remains in the nucleus and is degraded.
- there may be several different splicing patterns form a single transcript that can result in different mRNAs and thus different proteins.
- these alternative splicing patterns are very important in cell differentiation and embryonic development.
- tropomysin with 9 alternative splicing patterns: 4 fibroblast, 3 brain, 2 muscle (striated and smooth).
- average intron is approximately 2000b.
- the number of interons per gene is highly variable.
- extreme: Duchene's Muscular Dystrophy 79 exons and 78 introns spread over 2.5 million bases resulting in a functional mRNA of only 14,000 bases.
- in 1981 T. Cech described genes with self splicing interons (rRNA gene of a protozoan).
snRPs and scRPs
snRPs: small nuclear ribonuclear particle, this is a class of RNAs found in the nucleus that are bound to proteins (splicosome).
scRPs: small cytoplasmic ribonuclear particle, a class of RNAs found in the cytoplasm.
BOTH may function in control of genes.
STABILITY OF mRNA
- in prokaryotes most mRNAs only survive a few minutes and are rapidly degraded.
- in eukaryotes the mRNAs are more stable generally surviving 3 to 24 hours. There are a few specialized situations where they can survive for several days (hemoglobin).
- function as carrier molecules to move amino acids into line in response to the RNA sequence of the mRNA.
- at least 40 different tRNAs each is produced by a specific tRNA gene.
- - prokaryotes one copy of each tRNA gene.
- - eukaryotes multiple copies of each tRNA gene.
- generally rather small molecules--73 to 93 total bases.
- considerable modification of the transcript to incorporate several unusual bases.
- lots of intrastrand pairing. (20-21 bp established).
- all tRNAs have the same basic structure due to the characteristic pattern of intrastrand pairing.
- there are 14 bases which are invariable in all tRNAs.
LOADING OF AN AMINO ACID
- two important enzymes:
- - activating enzyme.
- - amino acyl -- tRNA synthetase. (20 different fxn enzymes)
- two step pocess requiring ATP for energy source.
LOADING AN AMINO ACID
amino acid + ATP + tRNA---------> [catalyst: aminoacyl tRNA synthetase]-------> aminoacyl tRNA + AMP
- 1. activating enzyme.
- 2. amino acyl tRNA synthetase.
acyl bond is high energy which provides the energy to drive the formation of the peptide bond.
- the 3 bases in the middle of the anticodon loop provide the specificity to the molecule.
- 3 bases of the codon H-bond specifically with the three bases of the anticodon.
THE GENETIC CODE
- based on a triplet code: three bases are required to specify a single amino acid.
- each triplet is called a codon an consists of three bases in a row that are read as a unit.
WHY A TRIPLET?
- since there are 20 different amino acids and only 4 bases in DNA/RNA a triplet is the smallest number that will allow unique specification of all 20 amino acids.
- (4)1 = 4 only 4 unique specifications.
- (4)2 = 16 only 16 unique specifications.
- (4)3 = 64 64 unique specifications.
DEGENERATIVE NATURE OF THE CODE
- since there are 64 different combinations the code must be "degenerative" in that there are several codons that can specify the same amino acid.
- extensive experimentation supported the triplet nature and "cracked" the code.
- due to the chemical polarity of both RNA and proteins the molecules that result from codon complementation produce linear molecules with known polarity.
- mRNA 5' ------------------ 3'
- protein NH2 ----------------- COOH
- due to the degenerative nature of the code Crick put forth the hypothesis.
- the firs two bases of a codon bind in very specific complementation while the third base is less confined and can physically wobble around to H-bond as best possible.
WOBBLE ON THE MOLECULAR LEVEL
WHAT IS I?
- I is the rare organic base ionisine.
- Inosine is commonly found in the anticodon of various tRNAs.
- due to it's bonding flexibility I in the third position (5') of the anticodon allows considerable wobble.
TRIPLET COON AND WOBBLE
completely degenerative: if the first two bases define the amino acid to be placed (third base has no effect).
partially degenerative: if the first two baes along with the presence of a purine versus a pyrimidine define different amino acids.
UNIVERSALITY OF THE CODE
- the genetic code is generally identical in all organisms with the exception of eukaryotic mitochondria.
- in the mitochondria:
- CUA: theronine instead of leucine.
- AUA: methionine instead of isoleucine.
- UGA: tryptophan instead of STOP.
TYPES OF AMINO ACIDS
- classified according to polarity of the molecule.
- polar -- hydrophylic.
- neutral -- neither hydrophobic or hydrophylic.
- nonpolar -- hydrophobic.
POLARITY AND PROTEIN SHAPE
- the polarity of the amino acid determines the general area that the unit will occur in the final folding of the protein.
- polar -- outside of protein.
- nonpolar -- inside of protein.
- neutral -- inside or outside of protein.
MUTATION AND THE GENETIC CODE
- obviously changes in the base sequence of the DNA or RNA result in changes in the amino acid coded for.
- changes in the second position tend to be of greater consequence than changes in the first or third position.
- changes in the first base change amino acid, but not the general type of amino acid.
- changes in the second base tend to change the general type of the amino acid and thus alter folding.
- changes in third base of minor effect due to degenerative nature and wobble.
- 1. chromosomal mutations.
- 2. point mutations.
molecular mutations which alter a single gene.
vast majority of mutations are recessive.
SOURCE OF MUTATION
- spontaneous occurrence.
- induced mutations.
- - mutations that can be traced to a specific source.
- - chemical or radiation based.
the spontaneous mutation rate varies with the organism and the gene under investigation.
bacteria: # of mutations / cell division.
eukaryotes: # of mutations / gamete / generation.
- the "general mutation rate" is 1x10-6.
- hot spots.
- cold spots.
ACCUMULATION OF MUTATIONS.
- assuming some mutations in each cell division a complex organism will accumulate many mutations over time.
- effectively each organism becomes unique.
- the important ones are the mutations passed through the gametes into the next generation.
- postadaptive mutation: mutate in response to a specific environmental factor or need. Lamarckian Evolution.
- preadaptive mutation: mutation occcurs BEFORE some environmental factor or need comes along and only the mutant types can exploit the new opportunity.
- forward mutation: wild to new mutant form.
- reverse mutation: mutant to wild type.
- suppressor mutation: second site mutation; mutant to wild.
- actually requires two separate mutations.
- intragenic: two different mutations within one cistron.
- intergenic: two different mutations in two different genes (often times a tRNA gene).
EFFECT BASED CLASSIFICATION
- morphological: visible in phenotype.
- biochemical: chemical basis understood.
- lethal: causes death of organism.
- semilethal: lower viability.
- conditional: expressed in specific environment.
POINT OR MOLECULAR MUTATIONS
- base substitutions: change one base for another base.
- frameshift: shift in reading frame due to start codon.
TWO TYPES OF BASE SUBSTITUTIONS
- missense mutations: the meaning or sense of the message has been changed (wrong amino acid).
- nonsense mutations: change from an amino acid coding codon to a stop codon. (truncated protein).
SOURCE OF BASE SUBSTITUTIONS
- tautomers: rare forms of the normal bases which result in altered H-bonding patterns.
- -keto forms are the normal forms.
- -imino and enol forms are rare (pair incorrectly).
MISSENSE MUTATION TYPES
- -purine substituted for the other purine.
- -pyrimidine substituted for the other pyrimidine.
- -pyrimidine substituted for a purine.
- -purine substituted for a pyrimidine.
- result from the loss or gain of one or more nucleotides.
- effect is due to the fixed reading frame after the AUG start codon.
- sever effects since this type effects each codon after the point of the mutation.
CLASSIFICATION OF MUTATION CAUSING AGENTS
- mutagenic: causing changes in the DNA.
- clastogenic: causing chromosome breakage and abnormalities.
- carcinogenic: causing development of cancer.
- teratogenic: causing developmental or birth defects.
- base analogs: mimic normal bases and get incorporated by mistake.
- direct chemical activity: change the base via chemical reaction.
- alkylating agents: add groups to existing bases in the DNA.
- acridine dyes: bind to DNA and block positions.
- chemicals that can substitute for a normal nucleobase in nucleic acids.
- cause transitions.
- ex. 5 Bromouracil, 5 Dromodeoxyurdine, 2 Aminopurine.
- removes amine groups (NH2).
- produces transitions.
- A to hypoxanthine A-T to H-C
- acts on cytosine.
- produces transitions.
- C-G to A-T.
- provides the right distance for them to fit inside DNA.
- once they bind, they won't let go.
- helicase isn't able to separate them.
- they are intercalated into DNA.
MUTATION VIA RADIATION
- ultraviolet radiation--"sunburn": wavelength of 2600Å strongly absorbed by DNA luckily there is poor penetration of this wavelength problems are thus limited to the skin.
- forms pyrimidine dimers: mostly T=T, but also C=C and C=T.
- many T=T dimers formed every day.
- very good repair mechanisms to deal with these common mutations. Photoreactivation using 4000Å wavelength.
- only a problem when over exposure occurs.
IONIZING OR PARTICLE RADIATION
- x-rays and the result of radioactive decay.
- deep penetration and many harmful effects due to basis in physical collision of molecules.
- target theory of damage: short exposure for a long time has the same effect as long exposure for a short time.
- actually a misnomer.
- really the genes DO NOT PROMOTE mutation, just less repair of normal mutations.
- genes are involved in DNA replication, recombination or repair.
PREVENTION OF MUTATION
- detoxification of damaging compounds especially superoxide radicals.
- superoxide dismutase converts superoxides to hydrogen peroxide.
- the hydrogen peroxide is then converted to water by a catalyase.
- also the mutT gene which prevents formation of 8-oxodoG (GO).
- direct reversal systems.
- excision repair systems.
- mismatch repair.
- post replication-recombination repair.
- SOS system.
DIRECT REVERSAL: PHOTOREACTIVATING ENZYME
- photoreactivating enzyme: photolyase binds to pyrimidine dimers and splits them apart in the presence of 4000Å wavelength of light.
DIRECT REVERSAL: ALKYLTRANSFERASES (METHYL TRANSFERASE)
enzyme removes alkyl groups from the O off the #6 position of guanine.
GENERALIZED EXCISION REPAIR
- common theme is cut out the bad bases and replace them with new bases.
- requires an excinuclease to cut out the nucleotides.
- the gap is filled in by repair synthesis.
- ligase seals the final phosphodiester bond to complete the strand.
SPECIFIC EXCISION REPAIR
- DNA Glycosylase:
- this enzyme cleaves the N-glycosidic bond (bond between the sugar and the base).
- the enzyme cuts out just the base which has been altered by mutation.
- leaves an apurinic or apyriminic site.
- final repair requires use of the AP endonucleases.
AP ENDONUCLEASE REPAIR
- repairs site which are without a base due to spontaneous loss or the action of the DNA gylcosylases.
- enzyme cleaves the phosphodiester bond at the AP site.
- followed by the activity of exonuclease, DNA polymerase I and DNA ligase.
GO SYSTEM REPAIR
- system works to prevent damage produced by the production of 8-oxodoG (GO) from Guanine.
- GO lesions are produced by oxidation.
- requires two different gylcolyases: mutM and mutY.
SHORT PATCH REPAIR
- classic excision repair system:
- - in prokaryotes cut out 12 or 13 bases.
- - in eukaryotes cut out 27 to 29 bases.
- in e. coli the excinucleases are:
- - uvrA recognizes damage and binds with uvrB to form a complex and lead uvrB to damage site.
- -uvrC then binds to the uvrB complex.
- each subunit makes a cut.
- the 12-mer is released by helicase and repaired by DNA pol I and DNA ligase.
LONG PATCH REPAIR
- long patch repair is very similar to short patch except in the length of the segment removed.
- generally 300+ bases are involved.
- repair of mismatched (noncomplementary) bases in the two strands.
- must be able to recognize mismatched bases.
- determine which is the incorrect base.
- excise the incorrect base and perform repair synthesis.
- recognition of correct is dependent on the postreplication methylation of DNA.
- methylation performed by adenine methylase A in GATC sequences (forms 6 methyl Adenine).
- thus the new strand (unmethylated) can be identified from the old strand (methylated).
POST REPLICATION - RECOMBINATION
- caused by a lesion which disrupts normal replication, causes the DNA Pol III to stop and restart after the lesion. end up with a single stranded gap.
- the gap is repaired by a piece of DNA cut from the sister molecule (specific recombination event using recA gene). good repair with few mistakes.
- this system is a general response to DNA damage which is multifactorial.
- basically an increased capacity to repair damaged DNA while inhibiting cell division.
- increases the number of copies of recA 50X.
- very error prone.
- transcriptionally active genes are preferentially repaired.
- three basic patterns of regulation:
- constitutive gene regulation
- inducible genes
- repressible genes
- these genes are constantly being produced.
- the mRNA is always present and producing the resulting protein.
- occurs in some bacteria. (prokaryotes)
- actually some variation due to different promoter strength.
INDUCIBLE AND REPRESSIBLE
- based on the need or demand for the gene products (enzymes).
- most are regulated at the point of transcription, but a few at translation.
- the production of the mRNA is dependent on the PRESENCE of a specific substance.
- "the molecule induces the system to turn on."
- works well for nutritional sources.
- the production of mRNA is dependent on the ABSENCE of a specific substance.
- "the molecule represses the system from production."
- good for the building blocks of the cell.
MUTATION AND GENE CONTROL
- polar mutation: mutations that result in messing up the genes that are downstream from the mutation site. (frameshift and nonsense).
- nonpolar mutation: mutations that have a localized effect and do not mess things up downstream. (missense).
BASIC IDEA OF CONTROL
control is achieved through opening and closing of the promoter site which thus prevents or allows the attachment of RNA polymerase holoenzyme.
- a group of genes (cistrons) which are under coordinate control.
- adjacent genes transcribed together.
- genes usually function in the same metabolic pathway.
- the genes that function in the break down of lactose into glucose and galactose.
- three cistrons:
- β-galactosidase Z (split)
- galactoside permease Y (get it into the cell fast)
- thiogalactoside acetylase A
OPERATOR SITE BLOCKING RNA POL
BASIC CONTROL OF THE LACTOSE OPERON
- differs due to a dual function controller.
- three genes to breakdown arabinose.
- still involves CAP and cAMP.
- loop formation prevent transcription.
- Dual function of C protein in control
- usually involved in the production of a major component of the cell. (amino acid synthesis).
- the end product is constantly needed in the cell.
- the genes are expressed unless there is an excess of the end product in the cell.
- five enzymes.
- forms tryptophan from chorismic acid.
- when tryptophan is present it binds with a repressor protein to block transcription at the operator region.
- dependent on the base sequence in the leader/attenuator region.
- first 130 bases of the functional mRNA.
- involves the production of a short (14 amino acid) leader polypeptide.
- leader polypeptide has two trp codons in a row.
- secondary structure of the leader/att. when termination occurs.
- results in the halting of transcription due to translational control.
EUKARYOTIC GENE REGULATION
gene control in eukaryotes is further complicated by:
- - embryological development.
- - tissue differentiation.
often the continuous on-off capabilities that we see in prokaryotes are not possible.
physiologic response is somewhat different.
THE PLAYERS IN EUKARYOTIC CONTROL
- trans acting factors: generally proteins that interact with the cis-acting elements.
- cis acting elements: DNA sequences that effect the control.
- complicated due to multifactorial and large distances involved.
cis acting (control) elements:
- promoter region
- promoter proximal elements
each can be recognized by some trans acting factors.
- -75 CCAAT/-23 "TATA like" sequence
- -meyers et al. determined that there are not sufficient signals in the promoter region to allow RNA polymerase to initiate transcription.
- these sequences and some additional (PPE) upstream bind proteins that enable RNA polymerase to bind and initiate transcription.
- -area of high GC content usually -200 to -100.
TAFs that bind at promoter and PPE are always available and are constitutively expressed.
- very similar to promoters, but capable of acting over large distance (up to 50 Kb). may be located either upstream on downstream.
- very intricate structure; two basic domains made up of five sequence elements with recognition sites for various TAFs.
TRANS ACTING FACTORS
- many different TAFs are known; some bind at the promoter region and some bind at the enhancer regions.
- all function to bind RNA polymerase II to DNA.
- the TAFs form a preinitiation complex which leads to the assembly of the initiation complex.
TAFs ALL THE "TATA-like" REGION
- at least eight TAFs involved in the binding of the "TATA-like"region of the promoter.
- TF-II and TBP are the basal transcription factors. minimal requirements for RNA polymerase binding.
DOMAINS OF TAF PROTEINS
TAF protiens with two separate domains:
1- a domain that recognizes and binds to a specific DNA sequences.
2- a domain that function in the activation of transcription.
DNA BINDING DOMAINS
- Helix-Turn-HelixHelix 1 and 2 contact other proteins.
- Helix 3 recognizes and binds to the DNA.
- Very similar to many bacterial regulator proteins.
DNA BINDING DOMAINS
- Zinc fingercystine and histidine rich area that complexes with zinc.
- protrusions of this complex resemble a finger.
- common in many mRNA promoters.
DNA BINDING DOMAINS
- Leucine zipperproteins that form dimers due to a hydrophobic interface formed by leucine spaced 7 amino acids apart.
- common in cancer genes.
DNA BINDING DOMAINS
- Helix-Loop-Helixalso function through dimeric protein interaction.
- two helices linked by a loop.
- occurs in some cancer and differentiation genes.
MOLECULAR MECHANISM OF CONTROL
- - basal transcription factors: required for transcription, but no effect on rate.
- - activators: increase rate of transcription.
- - repressors: decrease rate of transcription.
- - coactivators: function to communicate between the activators/repressors and the BTF.
- thought to involve looping of the DNA.
REGULATION OF TAFs
- many TAFs are activated by hormones, especially steroid hormones.
- - actually enter the cell and bind to specific TAFs in the nucleus.
- - function similar to inducer molecules in prokaryotes.
- 5 methyl cytosine regulation: active genes free of methyl groups.
- some TAFs assist in the recruitment of RNA Polymerase or other cofactors associated with transcription.
- some TAFs may function in chromatin remodeling which can change the pattern and position of nucleosomes.