genetics exam 4

cell theory

• living organisms are composed of one or more cells

• cell is a basic unit of life of the structural organization of an organism

• cell arrive from pre-existing cells and are NOT spontaneously generated

protobiont

• a precursor to living cells

• favor the idea that rna was the first macromolecule found in protobionts 

• consisted of an aggregate of molecules and macromolecules that acquired a boundary

• maintained an internal chemical environment distinct from that of its surroundings

rna world hypothesis

• rna can perform both info storage and enzymatic activity

• rna is involved in each of the major steps of gene expression

• maybe rna predates both dna and proteins

noncoding rna

• genes that don’t encode polypeptides

• binds to different types of molecules

• form different structures via intermolecular base pairing

• rna molecules can form stem-loop structures which may bind to pockets on the surface of proteins

long non coding rna

• longer than 200 nucleotides

• mis regulation of lcRNAs are is involved in many diseases

small regulatory rna (short ncRNA)

• shorter than 200 nucleotides 

• microRNA

ribozyme

• ncRNA molecules with catalytic function

• RNA enzyme/catalytic RNA

scaffold

• ncRNA binds a group of proteins at multiple binding sites

guide

• ncRNA binds to a protein and guides it to a specific site in the cell

• use base-pairing to direct proteins to specific locations (CRISPR)

decoy

• ncRNA recognizes another ncRNA and sequesters it 

• provides an alternate binding site for an inhibitory miRNA 

• miRNA normally binds to mRNA inhibiting translation

• decoy binds to the miRNA preventing binding to the mRNA 

blocker

• ncRNA physically prevents or blocks a cellular process from happening

• translation is repressed by ncRNA called micF, which does not code for a protein and is complementary to the to-be-translated gene

rRNA large subunit 

• ribozyme catalyzes peptide bond formation

RNase P

• a ribozyme endonuclease that cuts the 5’ end of precursor tRNAs to the correct position

small nucleolar RNAs (snoRNAs) 

• found in high amounts in the nucleolus 

• synthesis of rRNAs and the assembly of ribosomal subunits occurs in nucleolus 

• guide enzymes to covalently modify rRNAs in important locations 

• methylation of ribose on the 2’ hydroxyl group

• conversion of uracil to pseudouracil

snoRNAs act as scaffolds for modification proteins

• C/D box snoRNA methylation of ribose

• H/ACA box snoRNA converts uracil to pseudouracil

• scaffold function of snoRNAs to create snoRNPs

snoRNAs act as guides

• guide function of snoRNAs

• use base pairing to bring the modifying enzymes to the correct location on the rRNA

sense vs antisense rna 

• antisense rna: complementary to the mrna 

• sense rna: the mrna 

DsrA

• trans-acting ncRNA that can positively and negatively regulate translation in bacteria

• inhibits hns (histone-like nucleoid structuring protein) by blocking the RBS (ribosome biding site/shine-dalgarno) and part of DsrA is antisense

• activates rpoS (alternative sigma factor) by binding to the part of the rpoS mRNA that is complementary to the RBS (thus freeing it from stem-loop)

DsrA is complementary to multiple mRNAs

• different parts of DsrA have complementarity base-pairs to the hns or the rpoS mRNAs

• DsrA also has complementarity to other mRNAs

• the same sRNA can affect expression of multiple genes

HOTAIR (Hox transcript antisense intergenic RNA)

• recently discovered ncRNA alter chromatin structure

• HoxC genes act as a scaffold that guides two histone-modifying complexes to their target genes

mechanism of HOTAIR transcriptional repression

• Scaffold function binds:

  • PRC2 (Polycomb Repressive Complex 2)

    • Repressive – Adds trimethylation to histone H3K27

  •  LSD1 (Lysine Specific Demethylase 1)

    • Repressive – Removes methyl groups from H3K4 (Histone 3, lysine 4. Makes Histone 3 more positively charged)

• Guide function:

  • Base-pairing with GA-rich regions on the chromosome brings the scaffold to the appropriate location

  • Represses/Silences transcription

HOTAIR and cancer

• HOTAIR overexpression is implicated in many cancers 

discovery of RNAi

• hypothesis: inject antisense RNA into organisms to inhibit mRNA translation by complementary base-pairing

• this worked! and the effects of antisense rna persisted for a long time

• used a technique called FISH

• make sense and antisense mex3 RNA by in vitro transcription

• mixing in vitro synthesized sense and antisense RNA before injection allowed them to base-pair and form double stranded RNA (dsRNA)

FISH: Fluorescent in situ Hybridization

• use a probe DNA (or RNA) that is labelled fluorescently

• add probe to cells and allow it to hybridize to complementary sequences via base-pairing

• detect fluorescence by microscopy

miRNA (microrna)

• endogenous encoded by genes in eukaryotic organisms

• encode in the genome

• miRNA genes do not encode a protein

• give rise to small RNA molecules, typically 21 to 23 nucleotides

• not usually a perfect match to mRNAs

• act as guide ncRNA

siRNAs (short-interfering RNAs)

• exogenous encoded by foreign/invading genes (virus)

• foreign rna

• usually a perfect match or close to a perfect match to specific mRNAs

• act as guide ncRNA

Drosha 

• RNase located in the nucleus 

• cleaves pri-miRNAs into pre-miRNAs

dicer

• multisubunit complex RNase

• cleaves pre-miRNAs and pre-siRNAs into 20-25 bp miRNAs and siRNAs

RISC (rna inducing silencing complex)

• RNase

• gets rid of one RNA strand

• argonaute - RNase component of RISC

mechanism of RNA interference (siRNA)

• dicer 

• RISC/argonaute 

• perfect base pairing 

• target rna cleavage and protects against viral dna 

mechanism of RNA interference (miRNA)

• drosha

• dicer

• RISC/argonaute

• imperfect base-pairing

• translational inhibition or RNA degradation or p body localization

functions and benefits of RNA interference

• miRNA: important form of gene regulation; production of miRNAs silences the expression of specific mRNAs

• siRNA: provide a defense against viruses 

PIWI-interacting RNA

• found in animals

• ncRNA interacts with PIWI proteins and inhibits the movement of transposable elements

CRISPR-Cas

• defense against bacteriophages, plasmids and transposons

• ncRNAs play a key role

ncRNAs Called piRNAs Interact with PIWI Proteins

• transposable elements: segments of DNA that can become integrated into chromosomes 

• if TE is inserted into a genes, the event is likely to inactivate the gene

different transposition mechanisms of transposable elements

• simple transposition: cut and paste info, preserves or increases the number of transposons

• retrotransposition: goes thru an RNA intermediate, duplicates number of transposons

transposable elements influences on mutation and evolution

• TEs exist because they simply can!

  • They survive as long as they do not harm the host “selfish DNA”

• TEs exist because they offer some advantage

  • Bacterial TEs carry antibiotic-resistance genes

  • TEs may cause greater genetic variability through recombination

  • TEs may cause the insertion of exons into the coding sequences of structural genes

  • This phenomenon, called exon shuffling, may lead to the evolution of genes with more diverse functions

Purpose of PIWI RNAs & Proteins:

• prevent transposition-induced mutations from being passed on to the next generation