RNA Synthesis And Transport in Eukaryotes • Method: Pulse-Chase Labeling • At first, labeled RNA is exclusively in the nucleus. • Later, the labeled RNA is found in the cytoplasm.
Modifications to Eukaryotic pre-mRNAs • A 7-Methyl guanosine cap is added to the 5’ end of the primary transcript by a 5’-5’ phosphate linkage. • A poly(A) tail (a 20-200 nucleotide polyadenosine tract) is added to the 3’ end of the transcript. The 3’ end is generated by cleavage rather than by termination. • When present, intron sequences are spliced out of the transcript.
Eukaryotes Have Three RNA Polymerases Pol II is the only Polymerase that is routinely studied. Pol I and Pol III are very complicated.
What does the word Promoter mean? It is the place at which RNA Pol II binds. But the word is incorrectly used to describe Enhancers plus Promoter.
TFIID recognition site is TATAA How often is this site found in the genome? 1/45 Once every 1000 nucleotides 109 nucleotides or 106 times
Transient transfection More Cells But on a per cell Basis expression levels of -gal is about the same
The 3’ Poly(A) Tail AAUAAA
Interrupted Genes in Eukaryotes: Exons and Introns Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons. The introns are excised from the RNA transcripts prior to their transport to the cytoplasm.
Removal of Intron Sequences by RNA Splicing The noncoding introns are excised from gene transcripts by several different mechanisms.
Splicing • Removal of introns must be very precise. • Conserved sequences for removal of the introns of nuclear mRNA genes are minimal. • Dinucleotide sequences at the 5’ and 3’ ends of introns. • An A residue about 30 nucleotides upstream from the 3’ splice site is needed for lariat formation.
Types of Intron Excision • The introns of tRNA precursors are excised by precise endonucleolytic cleavage and ligation reactions catalyzed by special splicing endonuclease and ligase activities. • The introns of nuclear pre-mRNA (hnRNA) transcripts are spliced out in two-step reactions carried out by spliceosomes.
The Spliceosome • Five snRNAs: U1, U2, U4, U5, and U6 • Some snRNAs associate with proteins to form snRNAs (small nuclear ribonucleoproteins)
Logo for a) Splice acceptor b) Splice Donor c) Initiator Met
AG/GT CAG/NT exon 1 intron 1 exon 2 4 3 2 1 1 2 3 4 5 6 9 8 7 6 5 4 3 2 1 1 2
Protein Structure Proteins are complex macromolecules composed of 20 (?) different amino acids.
Amino Acids • Proteins are made of polypeptides. • A polypeptide is a long chain of amino acids. • Amino acids have a free amino group, a free carboxyl group, and a side group (R).
Peptide Bonds • Amino acids are joined by peptide bonds. • The carboxyl group of one amino acid is covalently attached to the amino group of the next amino acid.
Protein Synthesis: Translation The genetic information in mRNA molecules is translated into the amino acid sequences of polypeptides according to the specifications of the genetic code.
The Macromolecules of Translation • Polypeptides and rRNA molecules Euk: 28S, 18S, 5.8S, 5S • Amino-acid Activating Enzymes • tRNA Molecules • Soluble proteins involved in polypeptide chain initiation, elongation, and termination
Ribosomes What does “S” mean? Why do sizes get bigger?
The Nucleolus • In eukaryotes, the nucleolus is the site of rRNA synthesis and ribosome assembly
Synthesis and Processing of the 30S rRNA Precursor in E. coli ProKaryote numbers
rRNA Genes • rRNA Genes in E. coli • Seven rRNA genes distributed among three sites on the chromosome • rRNA Genes in Eukaryotes • rRNA genes are present in hundreds to thousands of copies • The 5.8S-18S-28S rRNA genes are present in tandem arrays in the nucleolar organizer regions of the chromosomes. • The 5S rRNA genes are distributed over several chromosomes.
Transfer RNAs (tRNAs) • tRNAs are adapters between amino acids and the codons in mRNA molecules. • The anticodon of the tRNA base pairs with the codon of mRNA. • The amino acid is covalently attached to the 3’ end of the tRNA. • tRNAs often contain modified nucleosides.