Genetics Ch. 9

posttranslational modifications

(a) enzymatic cleavage may remove an amino acid, split a polyprotein, or activate a zymogen: - N-terminal Met removal - polyprotein processing - zymogen activation (b) addition of chemical constituents may alter protein structure, activity, or cellular location: - phosphorylation: addition of phosphate group to serine (Ser) - glycosylation: addition of sugar to threonine (Thr) - lipidation: addition of fatty acid to N-terminal glycine (Gly) -ubiquitination: addition of ubiquitin (Ub) peptides to lysine (Lys)

nonsense suppression

(a) nonsense mutation causes incomplete nonfxna/truncated polypeptide (1st mutation) (b) nonsense-suppressing mutation = 2nd mutation -> allows for addition of amino acid & production of full length polypeptide: • due to 2nd mutation in tRNA anticodon that incorporates an amino acid rather than stopping translation at UAG

RNA splicing must be very very specific!!

- 1st cut @ splice donor site: • forms lariat intermediate w/ unique 2'-5' PD bond - 2nd cut at splice acceptor site generates free lariat: degraded - 2 exons are joined precisely

tRNAs

- 1° structure: nt seq - 2° structure: cloverleaf: • anticodon: complementary to codon of mRNA • antiparallel: 3'-CCU-5' -> tRNA anticodon; 5'-GGA-3' -> mRNA codon • 5'-3' structure: 1. D-loop (dihyrouridine-UH2) 2. anticodon loop 3. variable loop: varies in length, size of stem/loop 4. ψ-loop (pseudouridine) 5. acceptor stem = 3'end: always CCA - tertiary structure: L-shaped: • 1 end = anticodon loop • other end = acceptor stem

translation initiation: euks

- 40S subunit bind 5' mG cap then migrates down 5' UTR to 1st AUG (initiation codon) - adds tRNA w/ unmodified Met (tRNAi^Met) - then 60S joins; tRNAiMet in P-site: • initiation factors play role in assembly of initiation complex

gene's nt seq is colinear w/ amino acid sequence of encoded polypeptide

- Charley Yanofsky, 1960s trp⁻ auxotrophic (inability of an organism to synthesize a particular organic compound required for its growth) mutants in E. coli: • TrpA encodes: subunit of tryptophan synthetase • determined amino acid seq of mutant TrpA - observation: each pt mutation affects only 1 amino acid - conclusion: each nt is part of only 1 codon

central dogma

- DNA -> RNA = transcription: produces a mRNA transcript: • proks used directly • euks transcript is processed - RNA -> protein = translation: • occurs on ribosomes: made of protein & rRNA - depends on: 1. genetic code: decodes mRNA -> protein 2. tRNAs: specific amino acids placed @ correct position in growing polypeptide chain; have seq complementary to mRNA • doesn't explain all genes: some transcribed to nontranslated RNAs = rRNAs, tRNAs, etc

cracking the genetic code w/ mini-mRNAs

- Nirenberg and Leder (1965) - in vitro translation with synthetic trinucleotide mRNAs - tRNAs charged w/ amino acids, but only 1 amino acid is radioactive: • charged tRNA forms complex w/ ribosome & can't go through filter: CUC codes for LEU

transcription: DNA to RNA

- RNA polymerase -> transcription - promoters = DNA sequences = RNA polymerase binds to start transcription - RNA polymerase adds nts in 5'-to-3' direction from 1 template strand of DNA: • forms PD bonds using ribonucleotide triphosphates (ATP, CTP, GTP, UTP) • hydrolysis of bonds in NTPs provides energy for transcription • 3 Phase: initiation, elongation, termination - terminators = RNA sequences = signal to RNA polymerase to stop transcription

transcription initiation: bacteria

- RNA polymerase recognizes, binds to promoter near beginning of gene: • RNA holoenzyme = core RNA pol + σ (sigma) factor: σ factor = ⇧ affinity of RNA pol for promoter -> RNApol binds more tightly to DNA = closed promoter complex - DNA unwinds: template explosed = open promoter complex: • RNA pol chooses beginning 2 nucleotides: 5' end of mRNA by H-bond, complementary bping • phosphodiester bond formed betxn first 2 nts, sigma factor released = end of initiation • ↓ affinity of core RNA pol for promoter: remains attached but moves down template DNA

translation termination

- UAG, UAA, UGA: stop/nonsense codons: • NO tRNAs for stop codons - release factors (RFs): recognize & bind termination codons: • stops polypeptide synthesis • not as much known...: 1. tRNA of last amino acid releases completed polypeptide 2. tRNA & mRNA separate from ribosome 3. ribosome dissociates into large/small subunits

more gene structure

- all sequences in mature mRNA (all codons, UTRs: untranslated regions) except cap/tail transcribed by gene's exons - introns found at any location: • can even interrupt a codon • because of UTRs: start codon not always in 1st exon; stop codon not always in last exon

wobble: some tRNAs recognize more than 1 codon!!

- cells don't have 61 tRNAs or 1 tRNA per mRNA codon: • E.coli: 79 tRNAs w/ 42 different anticodons: 19 anticodons NOT represented - tRNAs can recognizes more than 1 codon for same amino acid - Crick: 3'nt (3rd/last) of codon NOT involved in specificity: • Gly: GGU, GGA, GGC, GGG = GGN • Gln: CAA, CAG = CAPu • His: CAU, CAC = CAPy - 5'nt of anticodon can pair w/ more than 1 type of nt in 3' position of codon: • codon -> 5' GGU 3' • anticodon -> 3' CCX 5' - 1 tRNA will recognize several/all codons for particular amino acid

translation elongation

- elongation factors (EF-proteins): bring appropriate tRNA to A-site (dpt on mRNA codon) - fMet from 1st tRNA in P site peptide bonded to amino acid in A site: • peptidyl transferase catalyzes peptide bond betxn 2 amino acids: tRNA in A-site = 2 amino acids on tRNA in A site! - ribosome slides to next codon & tRNA w/ dipeptide shifts from A-site -> P-site = translocation: • req EFs & energy (GTP) - empty tRNAfMet (no amino acid) exits from E-site of ribosome - empty A-site ready for next tRNA dpt on next codon - peptidyl transferase: dipeptide transferred to amino acid (Leu) on tRNA in A-site - translocation from A- to P-site; empty tRNA exits, repeat... - peptide grows N -> C-terminus as ribosome moves down mRNA 5' -> 3'

RNA processing: splicing: 1970s

- euk: DNA seq much longer than corresponding mRNAs: • mRNAs undergo internal processing - exons: "expressed genes": in both DNA & mRNA: • avg size ~ 200 bp - introns: "intervening regions": in DNA not mRNA: • avg size ~ 35 kb - introns removed, exons spliced together

Genomes

- gene expression: conversion of DNA to RNA & then decode RNA information to amino acid seq of a polypeptide - Central Dogma of Molecular Biology: proposed by Crick 1957

some loss-of-fxn alleles are dominant WT

- haploinsufficiency: one WT allele does NOT provide enough gene pdt - GLI3/GLI3+ humans have polydactyly: • transcription factor imperative for specification of digits • heterozygocity for loss-of-fxn mutation in GLI3 = dominant

gain-of-function mutations are usually dominant

- hypermorphic mutations generate more gene pdt or same amt of more efficient gene pdt: • FGFR3 gene encodes transmembrane Rc (Fibroblast GF Rc) that is usu active only when bound to FGF hormone to inhibits bone growth - Achondroplasia dominant hypermorphic allele of FGFR3^G480R: • altered Rc is constitutively active - neomorphic mutations: generate gene pdt w/ new fxn or that is expressed at inappropriate time or place - mutation in Antennapedia gene control/enhancer region of Drosophila causes ectopic (abnormal position) expression of leg-determining gene in cells that normally produce antennae

in euks, RNA processing after transcription

- in proks, primary transcript = mRNA - in euks, primary transcript is processed to make mature mRNA: 1. 5' methylated cap 2. 3' poly-A tail 3. introns removed by RNA splicing

identification of stop codons

- m gene encodes component of phage T4 capsule - Sydney Brenner found nonsense mutations (sense codon for amino acid is changed to a chain-terminating codon) = truncated polypeptides (results in shorter version of protein being produced): • point mutations (single nt) changed codon for amino acid to stop codon - ID'd UAG, UAA, and UGA as stop codons

transcription elongation

- making mRNA transcript by core RNA polymerase: • unwinds DNA as it moves = transcription bubble • extends mRNA in 5'-3' directions, on 3'-5' template • new RNA remains paired w/ template = DNA:RNA hybrid • behind bubble: DNA helix reforms -> displaces RNA - another RNA pol can initiate transcription when 1st off promotor; then another = multiple transcription bubbles; promoter -> end where transcripts are shortest

loss-of-fxn alleles are usually recessive

- null or amorphic mutations are alleles that completely block fxn of protein: • entire gene deletion - hypomorphic mutations produce less of WT protein or less effective protein - amt of xanthine dehydrogenase produced in flies w/ different genotypes - a^1 (null) & a^2 (hypomorphic) alleles are recessive to WT

RNA splicing

- occurs in/on splicesome = - 4 snRNPs = small nuclear ribonuclear proteins or 'snurps': • contain 1 or 2 of 5 snRNAs = U1, U2, U4, U5, U6: some snRNAs can bp w/ splice donor & acceptor sites to bring together 2 exon; ribozymes (ribonucleic acid enzymes) • ~50 proteins - exons: evolutionary building blocks?

RNA processing: 3' end

- poly A tail: euks - not coded by gene - 3' end = 100-200 As - added in 2 steps: 1. ribonuclease cleaves 3' end -> new 3' end: • cleavage signal = AAUAAA found 11-30nt upstream of where tail is added 2. polyA polymerase adds As to 3' end - fxn: polyA binding protein associates w/ 3' tail -> mRNA forms circle = stabilizes in cytoplasm; enhances translation initiation

transcription initiation in proks vs. euks

- prok genes promoters consensus sequences: • -35 = TTGACA • -10 = TATAAT (Pribnow box) - euk gene promoters: • RNA polymerase II transcribes protein coding genes • -25 = TATAA (TATA box) • often have enhancers: DNA sequences that can be 1000s bps away from promoter; req'd for efficient transcription • both common but not req'd

translation: pro vs. euk

- prok: • transcription & translation coupled b/c no nucleus; ribosomes can attach to 5' end of mRNA as it hangs off DNA template • initiation: 30S ribosome binding site in mRNA = Shine Delgarno sequence = AGGAGG near initiation AUG codon • many mRNAs polycistronic = 1 mRNA can code for several genes w/ several SD sequences, each translated independently • fMet: initiating amino acid - Euk: • nuclear membrane separates transcription from translation = not coupled/simultaneous • initiation: 40S ribosome binds 5'mG cap of mRNA, uses 1st AUG encountered in 5'UTR; 5' UTR = mG -> 1st AUG) • initiation @ single site, not polycistronic: each mRNA = 1 polypeptide • normal Met used as 1st amino acid

table 9.1: differences between prokaryotes and eukaryotes: translation

- prokaryotes: 1. unique initiator tRNA carries formylmethionine 2. mRNAs have multiple ribosome binding sites (RBSs) and can thus direct the synthesis of several different polypeptides 3. small ribosomal subunit immediately binds to the mRNA's ribosome binding site - eukaryotes: 1. initiator tRNA carries methionine 2. mRNAs have only one start site and can thus direct the synthesis of only one kind of polypeptide 3. small ribosomal subunit binds first to the methylated cap at the 5' end of the mature mRNA and then scans the mRNA to find the ribosomal bindings site

eukaryotic promoters are influenced by enhancers

- prokaryotes: • accessible promoter due to lack of chromatin (histone complex) - eukaryotes: • promoters must be cleared of chromatin • enhancers located far from promoter stabilize RNA polymerase and clear histones at promoter

chemical modifications of tRNAs are important for this fxn!

- read about selenocysteine (proks/euks) and pyrrolysine (proks): amino acid 21 & 22 - selenocysteine found in 25 human proteins; use UGA codons: p278

translation initiation: proks

- ribosome binding site in Proks for 30S ribosome: 1. Shine Delgarno (SD) box in mRNA: 5' AGG AGG 3': • 3' end of 16S rRNA complementary to SD (Shine Delagerno) seq 2. 5'-AUG-3' = initiation codon: • recognized by special: tRNAi^fMet w 5'-CAU-3' anticodon - 50S joins (30S + mRNA + tRNAi^fMet): tRNAi^fMet in P-site: • initiation factors play transient role in assembly of initiation complex

coding possibilities of synthetic mRNAs

- simple polynucleotides code for simple polypeptides - H.G. Khorana: mRNA w/ dinucleotide repeats: • UCU or CUC: which is LEU, which is SER?: UCU is SER & CUC is LEU

correlation of polarities of DNA, RNA, and polypeptides

- synthetic mRNA & in vitro translation: 5'-AAAUUU-3': • 5' end of mRNA = N-terminus of polypeptide • 3' end of mRNA = C-terminus - only 1 strand of DNA = template for mRNA = template strand or coding/sense strand - other strand RNA-like = same polarity & seq as RNA made. Or noncoding/nonsense strand - some of Khorana's RNAs (no polypeptide) stopped translation of polypeptide: • UAA, UAG, UGA don't correspond to an amino acid • stop or nonsense codons - good summary: p265

translation: mRNA to protein = tRNAs directs assembly of polypeptide on ribosome

- tRNAs: transfer RNAs: • adaptor molecules • transfer info from nucleic acid to protein - tRNA structure: • ssRNA, 74-95nt • contain modified bases • each carries 1 particular amino acid = charged tRNA • named = tRNA^Ala

eukaryotic RNAs are processed to form mRNA

- table 9.1 - euks require splicing of primary transcript due to presence of introns; proks have no introns - euks add 5' meG cap and 3' poly-A tail; proks do neither

transcription termination

- terminators = hairpin loops = specific mRNA seqs transcribed from DNA = signal end of transcription -> mRNA, RNA pol released from DNA: • intrinsic terminators: cause RNA pol to terminate on its own thru stem-loop • extrinic terminators: req additional proteins to terminate = rho termination - product of transcription = ssRNA: 1° transcript: • complementary to DNA betxn initiation & termination sites in template strand = same sequence as RNA-like strand (exc U for T)

alternative splicing

- usu 2 successive exon are joined but not always!: • can occur betxn splice donor site of 1 intron w/ splice acceptor site of diff downstream intron - particular exons are spliced out = alternative splicing - produces diff mRNA molecules ⇒ diff protein sequences (but partially overlapping): • membrane bound (B-lymphocytes) vs. secreted antibodies • ∵ (b/c) 28,000 genes can encode >100,000 proteins in humans

antimorphic (dominant negative) alleles prevent normal protein from fxning

- usually occurs in genes that encode multimeric proteins (multiple polypeptide chains) - mutant subunits block activity of normal subunits - kinky: a dominant-negative mutation in mice causing a kink in tail

translation: initiation, elongation, & termination

1. 1st translated codon: AUG towards 5' end, NOT directly @ 5' end of mRNA (remember 5'UTR...) 2. special initiating tRNA carries modified Met in proks: - formylmethionine (tRNAi^fMet) = recognizes initiation codon 3. ribosomes moves down mRNA: 5' -> 3' direction 4. amino acids added to C-terminus of growing polypeptide chain 5. translation terminates when ribosome reaches UAA, UAG, UGA (nonsense codons) @ 3' end of mRNA: - release factors -> release ribosome, polypeptide, mRNA: • 2-15 amino acids added/sec; faster in proks, slower in euks • 300 amino acid protein: 20" - 2.5'

RNA splicing must be very, very specific!!

3 sequences in 1° transcript: 1. splice donor site: 5' end of intron = PuPu | GUPuPu 2. splice acceptor site: 3' end of intron = Py12-14 AG | 3. branch site = CACUGAC; 5' end of intron attached to A: • forms lariat intermediate • 2nd cut generates free lariat: degraded • 2 exons are joined

experimental verification of the genetic code

C. Yanofsky: a) single-nt substitutions explain altered amino acids in trp⁻ -> trp⁺ revertants: • missense mutations: single nt substitutions that alter single amino acid b) amino acid changes due to proflavin-induced frame-shift mutations can be predicted: • alter reading frame

the genetic code: how do nts specify 20 amino acids?

a triple codon represents each amino acid: - 4^n, n = # of nt specifying an amino acid: • 1 nt specifies an amino acid = only 4 amino acid • 2 nt = 16 diff codons = 16 amino acids • 3 nt = 64 different triplet codons

some loss-of-fxn alleles can show incomplete dominance

incomplete dominance: phenotype varies continuously w/ amt of fxnal gene pdt

cracking the code: which codons represent which amino acids?

several breakthroughs in 1950s and 1960s: 1. incorporation of radioactive amino acids occurs in cytoplasm not nucleus: • must be intermediate made in nucleus, transported to cytoplasm 2. discovery of mRNA: radioactive uracil incorporated in nucleus (into RNA), some transported to cytoplasm: • RNA can base pair w/ DNA 3. in vitro translation of synthetic mRNAs: • new techniques to synthesize artificial RNAs with known nucleotide sequence • cellular extracts that allowed translation in test tube • = allowed synthesis of simple polypeptides

polyribosome

several ribosomes can translate same mRNA: many polypeptides made from 1 mRNA

the nucleus separates transcription from translation in eukaryotes

table 9.1: differences between prokaryotes and eukaryotes in of gene expression (part 1)

mutations classified by their effects on protein function

table 9.2

4 themes

1. base pairing: imperative for transfer info: - DNA -> RNA - RNA -> protein 2. polarities of DNA, RNA, protein molecules help guide mechanisms of gene expression: - mRNA that grows from its 5'-3' end on 3'-5' template DNA - mRNA translated 5'-3' - yields a protein running N-terminus - C-terminus 3. gene expression req energy input & specific enzymes at different points of process 4. mutations that change DNA information or obstruct flow of info can effect phenotype.

summary of the genetic code

1. genetic code has triplet codons 2. codons are nonoverlapping 3. three nonsense codons do not code for amino acids, but are stop codons: UAA, UAG, and UGA 4. genetic code is degenerate (multiple codons per amino acid: several codes have same meaning), yet unambiguous (one amino acid per codon) 5. reading frame is established from a fixed starting point: codon for translation initiation is AUG 6. mRNAs and polypeptides have corresponding polarities 7. mutations can be created in three ways: • frameshift: insertion or deletion of nucleotides in coding region resulting in altered sequence of amino acids at translation of codons • missense: produces an amino acid that is different from the usual amino acid at that position • nonsense: sense codon for amino acid is changed to a chain-terminating codon

ribosomes: sites of polypeptide synthesis

1. recognize mRNA 'start translation' signals 2. mediate/stabilize codon-anticodon interaction 3. enzyme activity of peptide bond formation - proks -> 70S: 3 rRNAs + 52 different proteins: 30S subunit & 50S subunit - euk -> 80S: 4 rRNAs & ~79 proteins: 40S subunit & 60S subunit - E.coli: • 30S: initially binds mRNA • 50S -> enzyme fxn: peptidyl transferase activity associate w/ 23S or 28S rRNA (catalyzes peptide bond formation) • both subunits together form: E site = exit site; P site = peptidyl site; A site = aminoacyl site

using synthetic mRNAs and in vitro translation to crack genetic code

1961: Marshall Nirenberg and Heinrich Mathaei: - added artificial mRNAs to cell-free (in vitro) translation systems

RNA processing: the 5' end

5' methylguanine cap: required for translation of mRNA -> protein in Euks: 1. backwards Guanine 2. linked to 5' end of mRNA by triphosphate bridge 3. not transcribed: • added by capping enzyme • methyl transferases add -CH3 to G & one or more of next nt • FXN: translation initiation factors to bind mRNA 5' cap

the genetic code is universal, almost!!

almost all cells alive today use the same genetic code: - mRNA from one organism can be injected into another organism's cells: • rabbit hemoglobin mRNA injected into frog eggs or cell-free wheat germ extracts ⇒ translated to protein = early evolution of code - some exceptions: • some species of ciliates: UAA & UAG (stop) ⇒ Gln • another ciliate: UGA (stop) = Cys • mitochondria (have own genome) of yeast: CUA (Leu) = Thr (mitochondria make only a few proteins; changes in code don't have a global affect) • certain proks: UAG = rare amino acid pyrrolysine • some proks/euks: UGA = rare amino acid selenocystine • bacteriophage: AUG or GUG: used as start in ~equal frequencies

mRNA codon bps w/ tRNA anticodon to direct incorporation of amino acids

amino acid of tRNA is NOT what determines incorporation: - change amino acid but not tRNA or anticodon: • tRNA^Cys -> tRNA^Ala now carries Ala - Ala incorporated whenever there is Cys codon in mRNA!

tRNA ligase

aminoacyl-tRNA synthetases: connects correct amino acid to specific tRNA: - very specific: must read both amino acid & anticodon - at least 1 aminoacyl-tRNA synthetase for each of 20 amino acids - 2 step process: 1. amino acid activation: ATP hydrolyzed -> AMP-amino acid = charged amino acid 2. charged amino acid transferred from AMP to 3'OH of tRNA acceptor stem CCA = charged tRNA

mutations & gene expression

mutations in amino acid-coding region generate a variety of issues: - silent mutations: has no effect on polypeptide made due to degeneracy (several codes have same meaning) of genetic code: • at which nt of codon would this usually occur? 3rd - missense mutations: • conservative: amino acid substitution w/ similar chemical properties; little or no effect on protein fxn • nonconservative: amino acid substitution with very different properties; more likely to affect polypeptide fxn - nonsense mutations: result in polypeptides smaller than WT (wild-type); truncated @ C-terminus - frameshift mutations: insertion/deletion can alter reading frame -> results in amino acid sequence unrelated to WT polypeptide - mutations outside coding region can alter gene expression: • promoter/enhancer: inhibit/prevent transcription • termination: inhibit amt of mRNA produced: less protein • start site mutations: no protein • splicing sites: no mature mRNA: no normal protein • ribosome binding site: ↓ ribosome binding = ↓ protein

mutations in genes encoding the components of gene expression have global effects

mutations in genes for rRNAs (polypeptide/protein synthesis), snRNAs (splicing), tRNAs (translation) that are transcribed but not translated (non-coding genes) may have large phenotypic effects: - transcription, mRNA splicing, translation, RNA processing: • usually lethal • mutations in tRNA genes can suppress mutations in protein-coding genes: nonsense suppressor tRNAs (READ ...)

sequence of a collagen gene, mRNA, and polypeptide

note: - RNA-like vs mRNA: • same seq • both 5'-3' - AUG in 2nd exon