REGULATION OF PROTEIN SYNTHESIS

II. Eukaryotes

Complexities of eukaryotic gene expression

! Several steps needed for synthesis of mRNA

! Separation in space of transcription and translation

! Compartmentation of proteins

Why is eukaryotic gene expression complex?

! Archaea provided genes for DNA metabolism, transcription, translation, DNA repair

! Bacteria provided genes for carbohydrate, amino acid, lipid metabolism

! Some bacterial genes persist in mitochondria, chloroplasts

Complexities of eukaryotic gene expression

! Epigenetics: which genes are expressed?

DNA methylation Histone modification siRNA gene silencing

! Several steps needed for synthesis of mRNA

Uncoiling of chromatin Remodeling of chromatin Transcription Transcript processing

DNA must uncoil to beused as a template

•Interphase vs mitosis•Differential expression as part of development

Evidence for the uncoiling of chromosomes

•“Lampbrush” chromosomes from Axilotl ova•Chromosome “puffs” in Drosophila salivary glands

Remodeling

• Removal of histones• Initiation of transcription

Transcription factors promote the binding ofRNA polymerase to template

! In eukaryotes, transcription isgenerally under positive control(proteins promote, rather than inhibit,RNA polymerase binding to DNAtemplate).

! Transcription factors bind to sequences upstreamfrom gene (up to1000 base pairs or more beforepromoter).

! DNA bends to form transcription complex.

Control of genes requires specificcombination of transcription factors

! One gene, multiple factors! One factor, multiple genes

(SRE: “stress response element”)

Eukaryotic transcript RNA must be processed before use

! Remove introns ! Cap ! Attach poly-A sequences.

Exons are the sequencespreserved in the mRNA!

Eukaryotic transcript RNA must be processed before use

! Remove introns (and splice exons together): alternative splicing can produce different mRNAs from one transcript

Removal of eukaryotic introns involves RNA enzymes ! Some introns of pre-mRNAs (called Group I

introns) are self-removing

! Some introns are removed (and the adjoining exons “spliced” together) by spliceosomes, made of protein plus RNA, with the RNA identifying the intron-exon boundaries

Eukaryotic transcript RNA must be processed before use

! Remove introns ! Cap ! Attach poly-A sequences.

Eukaryotic transcript RNA may be broken down before it can be used

! siRNAs and miRNAs (small-interfering RNAs and micro RNAs) can regulate translation

! siRNAs and miRNAs are cut from

double-stranded RNA; one strand joins a protein complex

! The protein-siRNA complex breaks

down mRNAs that contain complementary sequences

! The protein-miRNA complex breaks

down mRNAs, or it binds to them, preventing translation

(Petunia plants expressing chalcone synthase antisense RNA)

Some proteins must be guided to theirdestination and processed.

! Eukaryotic cells have many compartments.! Leader sequences signal import into E.R.! Transit sequences direct transport into

mitochondria, plastids, nuclei.! Leader, transit sequences generally removed.

Turnover (breakdown) of proteins also controlstheir concentration in the cell

Sifers, Science, 9 July 2010, 154-155

A lack of proper breakdown of proteins causes a “conformational disease”---A new compound (CPZ) can help

Summary

Regulation of protein synthesis is necessary in allcells, but much more complex in eukaryotes, becauseboth the cells and the organism they form are morecomplex.

Uncoiling of chromatin: DNA, histone modification Remodeling of chromatin: removing histones Transcription: binding of transcription factors Transcript processing: intron removal, capping, poly-A addition Transcript turnover: siRNAs, miRNAs Translation: compartmentation Protein turnover