tRNA structure

What’re the 2 important components of tRNA structure?

1. Acceptor domain

2. Anti codon loop

Where does amino acids attach to?

• 3’ OH on as it extends further than 5’ end

What does the acceptor domain do?

• (3’-ACC) → amino acids is carried by the 3’ OH end of the tRNA

What does the anti-codon loop do?

• (central loop) → contains 3 bases (anti codon) which determines placement of a specific amino acid into the growing polypeptide chain

Explain what occurs due to the 3’ end having unpaired CCA

• Generally not encoded in the tRNA gene

• Added sequentially one at a time by CCA adding enzyme Using CTP and ATP as substrates

• Cognate amino acid covalently attached to A

What facilitates the linkage of correct amino acid to tRNA?

• Aminoacyl tRNA synthetase

• Requires specific contacts made between regions of the tRNA and the synthetase

Explain what occurs in step 1 of the binding of amino acid to 3’ CCA end of tRNA (mediated by aminoacyl tRNA synthetase)

1. Activation of amino acid by reaction with ATP

• Amino acid + ATP ←> aminoacyl- AMP + P-P (pyrophosphate PPi)

Aminoacyl intermediate formed remains bound to the tRNA synthetase until collision with the appropriate tRNA molecule

Explain what occurs in step 2 of the binding of amino acid to 3’ CCA end of tRNA

2. Activated amino acid is bonded to the CCA stem of its tRNA to form a charged tRNA

• Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

• Pyrophosphate (PPi) formed in first rxn is split into 2 molecules of inorganic phosphate cuz ATP used and AMP formed in these rxns, a total of 2 energy rich phosphate bonds are expended to charge a tRNA with its cognate amino acid

• Amino-acyl AMP is now bonded to CCA stem of its tRNA and exits synthetase

• Energy rich bond provides energy to incorporate amino acids into growing polypeptide chain

What does mRNA do?

• Carries information about protein structure

• ~90% of genes in E.coli code for mRNA (structural gene)

• MRNA has short life span

What are the 3 sections of mRNA?

1. Leader sequence

2. Coding sequence

3. Trailer sequence

What does leader sequence in mRNA do?

• On 5’ end of mRNA transcript

• Contains a specific ribosome binding site (shine dalgarno sequence)

What does coding sequence of mRNA do?

• Info is organized in codons (ie sets of 3 nucleotides)

• Each codon is complementary to an anticodon of a tRNA which specifies amino acid

• Generally begins with AUG start codon encoding chemically modified methionine called N-formal methionine

• Ends with stop (ie nonsense codon)

What does trailer sequence of mRNA do?

• Transcribed to make the 3’ end of mRNA

• Sequence of trailer determines stability of mRNA

  • Eg: degradation impeded by secondary structure, modulated by RNA binding proteins into response to environmental stress signals etc.

Explain the way the reading frame works

• It starts from the start codon, the mRNA transcript is read “in frame” with each frame corresponding to a codon (set of 3 nucleotides) with no interruption between frames

What’s a frame-shift mutation?

• Caused by addition of deletion of nucleotide in DNA sequence, when transcribed, can put the coding sequence “out of frame”

Explain what the polypeptide is affected by

• Affected due to amino acid sequence change thereby affecting shape and function of protein, or maybe truncated if a non-sense (stop) codon is generated

What are the 3 types of proteins covered?

1. Catalytic proteins

2. Structural proteins

3. Regulatory proteins

What do catalytic proteins do?

• Catalysts for chemical reactions that occur in the cell

What do structural proteins do?

• Integral parts of the major structures of the cell (membranes, walls, ribosomes, etc)

What do regulatory proteins do?

• Control cell processes by variety of mechanism including binding to DNA and affecting transcription (gene expression)

What are proteins polymers of?

Amino acids

An amino acid contains an amino group (NH2) and a carboxylic acid group (COOH) that are attached at the alpha carbon

What’s a peptide bond?

• Linkage between the COOH group of one amino acid with the amino nitrogen of the second amino acid with the loss of one water molecule

What’s a dipeptide?

• 2 amino acids bonded by peptide linkage

What’s a tripeptide?

• 3 amino acids bonded by 2 peptide linkages

What’s a polypeptide?

• Multiple peptide bond linked amino acids

Explain what happens as a result of a protein consisting of one or more polypeptides

• Translation of a mRNA transcript will result in a polypeptide sequence which may be the final protein product or may be part of a final protein product (subunit)

Explain what primary protein structure is

• Linear sequence of amino acids in a polypeptide chain that ultimately determines the folding pattern of the polypeptide, which in turn determines its biological activity

What affects primary protein structure?

• As little as an amino acid change can affect primary structure affecting function

Explain secondary structure of polypeptides

• Generated by hydrogen bonding between oxygen and nitrogen atoms of 2 peptide bonds to either form alpha helix or beta sheet

• A polypeptide can contain regions of alpha helix and regions of beta sheet

Explain tertiary structure of polypeptides

• Higher order of structures generated by interactions between the R groups of the amino acids in a polypeptide

• 3D form dependent of hydrophobic interactions, with lesser contributions from H bonds, ionic bonds and disulfide bonds

• Many proteins consist of 2 or more polypeptides (subunits) and thus show quaternary structure stabilized by various interactions and also by disulfide bonds formed between cysteine residues in 2 diff subunits