Transcription and RNA Processing

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.

Awful representation

Correct Representation of DNA

RNA make a new “top strand”

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.

miRNAproduction

A Typical RNA Polymerase II Promoter

What does the word Promoter mean?

It is the place at which RNA Pol II binds.But the word is incorrectly used to describeEnhancers plus Promoter.

Initiation by RNA Polymerase II

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

More CellsBut on a per cellBasis expression levels of -gal is about the same

Transient transfection

Stable transfection

The 7-Methyl Guanosine(7-MG) Cap

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.

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 fora) Splice acceptor

b) Splice Donor

c) Initiator Met

AG/GTAG/GT CAG/NTCAG/NT

exon 1 exon 1 intron 1 intron 1 exon 2exon 2

9 8 7 6 5 4 3 2 1 1 2 4 3 2 1 1 2 3 4 5 6

Chapter 12Translation 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?

RibosomesWhat 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

ProKaryotenumbers

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 InitiationChain ElongationChain Termination

Translation Initiation in E. coli

30S subunit of the ribosomeInitiator tRNA (tRNAMet)mRNAInitiation Factors IF-1, IF-2, and IF-3One molecule of GTP50S 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 completeOrderedNearly 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 sameRepertoire 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 tRNATyr gene, then could onehave 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 IITATAA

CCATGG (Nco I site and Kozak Rule)ATG

AGGT….spliceGT……………A………polypyrimidine AG

PolyA recog sequenceAATAAA

The Reasons why ATG is a single codonand TGG is a single codon.

To Know for Exam