Molecular Biology (CLS 354 )

DNA , replication, transcription, translation -2

Dr/ Abdul-Raouf

Translation - RibosomesTwo substances that play key role in translation:

ribosomsS & transfer RNA Ribosomes are the protein synthesizing machines

Ribosome subunits are designated with numbers such as 50S or 30S.

Number is the sedimentation coefficient - a measure of speed with which the particles sediment through a solution spun in an ultracentrifuge

Each ribosomal subunit contains RNA and protein.

The two ribosomal subunits both contain ribosomal RNA (rRNA) molecules and a variety of proteins rRNAs participate in protein synthesis but do NOT code for proteins No translation of rRNA occurs

(a) (b)

Figure shows E. coli ribosome structure.(a) The 70S ribosome is shown from the side with the 30S particle (yellow) and the 50S particle (red) fitting together.(b) The 70S ribosome is shown rotated 90 degrees relative to the view in part (a). The 30S particle (yellow) is in front, with the 50S particle (red) behind.

Summary Ribosomes are the

cells protein factories Bacteria contain 70S

ribosomes Each ribosome has 2

subunits 50 S & 30 S

Each subunit contains rRNA and many proteins

Figure shows Composition of the E. coli ribosome. The arrows at the top denote the dissociation of the 70S ribosome into its two subunits when magnesium ions are withdrawn. The lower arrowsshow the dissociation of each subunit into RNA and proteincomponents in response to the protein denaturant, urea. The masses(Mr, in daltons) of the ribosome and its components are given inparentheses.

Translation Adapter Molecule Generating protein from ribosomes requires change from the

nucleic acid to amino acid This change is described as translation from the nucleic acid

base pair language to the amino acid language Crick proposed that some type of adapter molecule was needed

to provide the bridge for translation, perhaps a small RNA

Transfer RNA: Adapter Molecule

Transfer RNA is a small RNA that recognizes both RNA and amino acids

A cloverleaf model is used to illustrate tRNA function

One end (top) binds amino acid with sequence specific to a particular amino acid

Bottom end contains a 3 base pair sequence that pairs with complementary 3-bp sequence in mRNA

Figure 3.17 Cloverleaf structure of yeast tRNAPhe.

At top is the acceptor stem (red), where the amino acid binds to the 3-terminaladenosine.

At left is the dihydro U loop (D-loop, blue), which contains at least one dihydrouracilbase.

At bottom is the anticodon loop(green), containing the anticodon.

The T-loop (right, gray) contains thevirtually invariant sequence TC.

Each loop is defined by a base pairedstem of the same color.

Codons and Anticodons Enzymes that catalyze attachment of amino acid to tRNA are

aminoacyl-tRNA synthetases

A triplet in mRNA is called a codon

The complementary sequence to a codon found in a tRNA is an anticodon.

Definition :

Codon : Three-base segment of mRNA that specifies amino acids.

Anticodon: 1. Three-base segment of tRNA that docks with a codon.

2. Docking results in deposition of amino acid.

Figure shows Codonanticodon recognition. The recognition between a codon in an mRNA and a corresponding anticodon in a tRNA obeys essentially the same WatsonCrick rules as apply to otherpolynucelotides.

Here, a 3AAGm5 anticodon (blue) on a tRNAPhe isrecognizing a 5UUC3 codon (red) for phenylalanine in an mRNA. The Gm denotes a methylated G, which base-pairs like an ordinary G.

Notice that the tRNA is pictured backwards (3 5) relave to normal convenon, which is 5 3, le to right. That was done to put its anticodon in the proper orientaon (3 5, le to right) to base-pairwith the codon, shown convenonally reading 5 3, left to right.

Remember that the two strands of DNA are antiparallel; this applies to any double-stranded polynucleotide, including one as small as the3-bp codonanticodon pair.

Initiation of Protein Synthesis The initiation codon (AUG) interacts with a special aminoacyl-

tRNA In eukaryotes this is methionyl-tRNA In bacteria it is a derivative called N-formylmethionyl-tRNA

Position of the AUG codon: At start of message AUG is initiator In middle of message AUG is regular methionine

Shine-Dalgarno sequence lies just upstream of the AUG, functions to attract ribosomes Unique to bacteria Eukaryotes have special cap on 5-end of mRNA

Translation Elongation

After initiation, initiating aminoacyl-tRNA binds to a site on the ribosome, P site

Elongation adds amino acids one at a time to the initiating amino acid

First elongation step is binding second aminoacyl-tRNA to another site on the ribosome, A site

This process requires: An elongation factor, EF-Tu Energy from GTP

Figure shows Summary of translation elongation. (a) EF-Tu, with help from GTP, transfers the

second aminoacyl-tRNA to the A site. (The P and A sites are conventionally represented on the left and right halves of the ribosome, as indicated at the top.)

(b) Peptidyl transferase, an integral part ofthe large rRNA in the 50S subunit, forms a peptide bond between fMet and the second aminoacyl-tRNA. This creates a dipeptidyl-tRNAin the A site.

(c) EF-G, with help from GTP, translocates the mRNA one codons length through the ribosome. This brings codon 2, along with thepeptidyl-tRNA to the P site, and codon 3 to the A site. It also moves the deacylated tRNA out of the P site into the E site (not shown), from which itis ejected. The A site is now ready to accept another aminoacyl-tRNA to begin another round of elongation.

Termination of Translation and mRNA Structure Three different codons (UAG, UAA, UGA) cause translation

termination Proteins called release factors recognize these stop codons causing

Translation to stop Release of the polypeptide chain

Initiation codon and termination codon at the ends define an open reading frame (ORF)

SUMMARY Translation elongation involves three steps: (1) transfer of the second aminoacy-tRNA to theA site;

(2) formation of a peptide bond between the first amino acid in the site and the second aminoacyl tRNA in the A site;

(3) translocation of the mRNA one codons length through the ribosome, bringing the newly formed peptidyl-tRNA to the P site.

Mutations Genes accumulate changes or mutations

Mutation is essential for evolution

If a nucleotide in a gene changes, likely a corresponding change will occur in an amino acid of that genes protein product If a mutation results in a different

codon for the same amino acid it is a silent mutation

Often a new amino acid is structurally similar to the old and the change is conservative

Sickle Cell Disease

Sickle cell disease is a genetic disorder

The disease results from a single base change in the gene for b-globin Altered base causes insertion an incorrect amino acid into

one position of the b-globin protein

Altered protein results in distortion of red blood cells under low-oxygen conditions

This disease illustrates that a change in a gene can cause corresponding change in the protein product of the gene