BIOL 442 Lecture 31

possible destinies for mRNA

• some are translated and then degraded

• some are stored in the cytoplasm for later use

• some are subject to post-transcriptional/translational control by miRNAs

RNAi (RNA interferene)

sequence-specific gene-silencing process that occurs post-transcriptionally

RNAi is triggered by

double stranded RNA (dsRNA)

classes of dsRNA

small interfering RNA (siRNA)

microRNA (miRNA)

siRNAs function via autosilencing

via autosilencing

siRNAs silence the same locus where they originate

siRNAs + defense

defend the genome

• x. a virus RNA triggers mechanism to cleave the viral RNA

• scientists can insert dsRNA into cell to experimentally inhibit gene expression

miRNAs

do heterosilencing

they are derived from miRNA genes are specify the silencing of protein-coding genes

how do mRNAs act as a mechanism for post-transcriptional control?

• they target mRNA for degradation or inhibition of translation

• they are made from gene transcripts and form hairpin structures

in the human genome miRNA genes

are mainly located in introns of protein-coding mRNAs

miRNA pathway in the nucleus

miRNA is made from miRNA genes via RNAP

pri-miRNA + Drosha

pri-miRNA folds into a 2’ hairpin structure

Drosha + cofactor snip off non-hairpin structure, making short hairpin RNA (shRNA)

miRNA pathway in cytoplasm

miRNA is processed by Dicer that snips off the top of the hair pin

miRNA + RISC Argonaute

double stranded miRNA is loaded into RISC loading complex and one strand is cast off by Argonaute “slicer”

RISC with miRNA association with mRNA

association via complementary base pairing between miRNA + mRNA

short complementary bp result in

repression of translaiton

extensive complementary base pairing

results in mRNA degradation

translation occurs in the cytoplasm if

mRNA is not subject to post-transcriptional/translational regulation

Ribosome = protein synthesis machinery

some are free in cytosol, some are bound in ER

Ribosome actions

decode the genetic code in mRNA

catalyze formation of peptide bonds between AA

ribosome components

large + small subunits, rRNAs, proteins

rRNA makes up catalytic center, decoding site, A, P, E sites + subunit interfaces

Fidelity of translation is dependent on

1.) aminoacyl-tRNA synthesis: is the correct AA added to make the charged tRNA

2.) codon-anticodon recogition

Elongation cycle A site

initial binding - tRNA brings AA to A site

check if bp codon-anticodon is correct, GTP hydrolyzed

P site

where peptide bond between AA is formed

translocation to E site

E site

uncharged tRNA is held until released, termination when stop codon reached

differences in ribosomes might explain human diversity

• link between rRNA mutations + height/weight

• rRNA variants are incorporated into ribosome + alter its structure