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.

Initiation by RNA Polymerase II

TFIID recognition site is TATAA How often is this site found in the genome? 1/4 5 Once every 1000 nucleotides 10 9 nucleotides or 10 6 times

More Cells But on a per cell Basis expression levels of  -gal is about the same Transient transfection

Stable transfection

Recruitment Model

The 7-Methyl Guanosine (7-MG) Cap

The 3’ Poly(A) Tail AATAAA

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.

Excision of Intron Sequences

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)

What are Logo plots?

Logo for a) Splice acceptor b) Splice Donor c) Initiator Met

AG/GTCAG/NT exon 1 intron 1 exon 2

Chapter 12 Translation and the Genetic Code

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

Why does one need the ribosome to translate mRNA?

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

Synthesis and Processing of the 45S rRNA Precursor in Mammals

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.

What is Inosine?

Inosine

tRNA Structure

Specificity of tRNAs  tRNA molecules must have the correct anticodon sequence.  tRNA molecules must be recognized by the correct aminoacyl-tRNA synthetase.  tRNA molecules must bind to the appropriate sites on the ribosomes.

Codon Specificity Resides in the tRNA, Not the Attached Amino Acid.

tRNA Binding Sites on the Ribosome (Ribosme moves like an enzyme)

Stages of Translation  Polypeptide Chain Initiation  Chain Elongation  Chain Termination

Translation Initiation in E. coli  30S subunit of the ribosome  Initiator tRNA (tRNA Met )  mRNA  Initiation Factors IF-1, IF-2, and IF-3  One molecule of GTP  50S subunit of the ribosome

The Shine-Dalgarno Sequence

Translation Initiation in Eukaryotes  The amino group of the methionine on the initiator tRNA is not formylated.  The initiation complex forms at the 5’ terminus of the mRNA, not at the Shine-Dalgarno/AUG translation start site.  The initiation complex scans the mRNA for an AUG initiation codon. Translation usually begins at the first AUG.  Kozak’s Rules describe the optimal sequence for efficient translation initiation in eukaryotes.

Polypeptide Chain Elongation  An aminoacyl-tRNA binds to the A site of the ribosome.  The growing polypeptide chain is transferred from the tRNA in the P site to the tRNA in the A site by the formation of a new peptide bond.  The ribosome translocates along the mRNA to position the next codon in the A site. At the same time, –The nascent polypeptide-tRNA is translocated from the A site to the P site. –The uncharged tRNA is translocated from the P site to the E site.

Elongation of Fibroin Polypeptides (A mRNA can have multiple Ribosomes

Polypeptide Chain Termination  Polypeptide chain termination occurs when a chain-termination codon (stop codon) enters the A site of the ribosome.  The stop codons are UAA, UAG, and UGA.  When a stop codon is encountered, a release factor binds to the A site.  A water molecule is added to the carboxyl terminus of the nascent polypeptide, causing termination.

No tRNA exists for stop codons!

Dissociation upon finish of protein synthesis

The Genetic Code The genetic code is a nonoverlapping code, with each amino acid plus polypeptide initiation and termination specified by RNA codons composed of three nucleotides.

Properties of the Genetic Code  The genetic code is composed of nucleotide triplets.  The genetic code is nonoverlapping. (?)  The genetic code is comma-free. (?)  The genetic code is degenerate. (yes)  The genetic code is ordered. (5’ to 3’)  The genetic code contains start and stop codons. (yes)  The genetic code is nearly universal. YES :)

A Triplet Code*

A Single-Base Pair Insertion Alters the Reading Frame*

A suppressor mutation restores the original reading frame.*

Insertion of 3 base pairs does not change the reading frame.*

Evidence of a Triplet Code: In Vitro Translation Studies  Trinucleotides were sufficient to stimulate specific binding of aminoacyl-tRNAs to ribosomes.  Chemically synthesized mRNAs containing repeated dinucleotide sequences directed the synthesis of copolymers with alternating amino acid sequences.  mRNAs with repeating trinucleotide sequences directed the synthesis of a mixture of three homopolymers.

Deciphering the Genetic Code

You must know single letter codes and some triplets!

The Genetic Code  Initiation and termination Codons –Initiation codon: AUG –Termination codons: UAA, UAG, UGA  Degeneracy: partial and complete  Ordered  Nearly Universal (exceptions: mitochondria and some protozoa)

Key Points  Each of the 20 amino acids in proteins is specified by one or more nucleotide triplets in mRNA. (20 amino acids refers to what is attached to the tRNAs!)  Of the 64 possible triplets, given the four bases in mRNA, 61 specify amino acids and 3 signal chain termination. (have no tRNAs!)

Key Points  The code is nonoverlapping, with each nucleotide part of a single codon, degenerate, with most amino acids specified by two to four codons, and ordered, with similar amino acids specified by related codons.  The genetic code is nearly universal; with minor exceptions, the 64 triplets have the same meaning in all organisms. (this is funny)

Do all cells/animals make the same Repertoire of tRNAs?

The Wobble Hypothesis: Base-Pairing Involving the Third Base of the Codon is Less Stringent.

Base-Pairing with Inosine at the Wobble Position

Suppressor Mutations  Some mutations in tRNA genes alter the anticodons and therefore the codons recognized by the mutant tRNAs.  These mutations were initially detected as suppressor mutations that suppressed the effects of other mutations.  Example: tRNA mutations that suppress amber mutations (UAG chain-termination mutations) in the coding sequence of genes.

Making a (UAG) Mutation

Translation of an amber (UAG) Mutation in the Absence of a Suppressor tRNA

Translation of an amber Mutation in the Presence of a Suppressor tRNA Note it is amber su3…why?????????

Translation of an amber Mutation in the Presence of a Suppressor tRNA If there was a single tRNA Tyr gene, then could one have a amber supressor of it?

Historical Comparisons  Comparison of the amino acid sequence of bacteriophage MS2 coat protein and the nucleotide sequence of the gene encoding the protein (Walter Fiers, 1972). Was this first????  Sickle-cell anemia: comparison of the sequence of the normal and sickle-cell alleles at the amino acid level and at the nucleotide level.

Are the proteins produced a pure reflection of the mRNA sequence???? tRNA environment, protein modifications post-translationally

RNApol II TATAA CCATGG (Nco I site and Kozak Rule) ATG AGGT….splice GT……………A………polypyrimidine AG PolyA recog sequence AATAAA The Reasons why ATG is a single codon and TGG is a single codon. To Know for Exam