PROTEIN SYNTHESIS

Molecular Genetics Strand

TRANSCRIPTION (in nucleus)

1. Polymerase Binding and Initiation - RNA Polymerase II binds to regions of DNA called promoters - a promoter is about 100 nucleotides long and is composed of

an initiation site and nucleotides that are “upstream” from the intitiation site (i.e. upstream TATA box)

- also need proteins called transcription factors to aid

polymerases in search for promoter region (i.e. RNA Polymerase II recognizes the transcription factor-

promoter region “complex”)

- once RNA Polymerase II binds to the promoter, another RNA Polymerase enzyme separates the DNA at the initiation site and transcription begins

- there are up to 40,000 RNA Polymerase molecules in the nucleus

2. Elongation - RNA Polymerase II moves along the DNA, untwists, and

separates strands exposing about 10 nucleotides at a time

- it adds RNA nucleotides to the 3’ end of the growing

RNA molecule - only one DNA strand is used as a template - it adds about 60 nucleotides /s and does not require a

primer - the promoter does not get transcribed

- an hnRNA (heterogeneous nuclear RNA) molecule peels away from the DNA template

- a single gene can be transcribed by a convoy of RNA Polymerase II molecules if the protein is required in a large amount

3. Termination

- transcription proceeds until RNA Polymerase II reaches a “termination” site on DNA

- the most common eukaryotic sequence is AATAAA

- there is no more addition of RNA nucleotides, the hnRNA is released, and polymerase is free to bind to another promoter

Animation 1

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RNA PROCESSING (in nucleus) - one transcript molecule (i.e. one hnRNA) represents one gene - hnRNA has a bunch of non-coding regions and must be modified into

mRNA (in eukaryotes) before it can leave the nucleus - what happens : • introns (non-coding regions) are cut out by enzymes called

spliceosomes which are made from snRNPs – small nuclear ribonucleoproteins

- snRNPs are made from snRNA (small nuclear RNA) • a 5’ guanosine triphosphate (GTP) “CAP” is added • a 3’ poly-adenine “TAIL” is added by poly-A polymerase - no quality control enzymes - mRNA now ready to move into the cytoplasm for translation

TRANSLATION (in cytoplasm)

- mRNA (the altered hnRNA) moves into cytoplasm - process of translating mRNA (language : nucleotides) into protein

(language : amino acids) - the translator is tRNA (transfer RNA) - tRNA transfers AA from the cytoplasmic pool to a ribosome, and the

ribosome adds AAs to the growing end of a polypeptide chain - tRNA can also travel from the nucleus to the cytoplasm - tRNAs can be used repeatedly

- tRNA is single-stranded, about 80 nucleotides long, and shows secondary structure (it has folds – a cloverleaf shape)

- it has an anticodon end and an acceptor site

- the anticodon is complementary to the mRNA codon

- there are only 45 tRNA molecules, although there are 61 AA codes

- some tRNAs have anticodons that can recognize 2 different codons because the rules for base pairing between the codon’s third base and the tRNA’s anticodon are not as strict as those for DNA and the mRNA codons (wobble)

• how does the tRNA get the AA joined to it?

- each tRNA is matched with the correct AA by a specific enzyme called an aminoacyl-tRNA synthetase

(there is one aa-tRNA synthetase for each AA)

- coordinating tRNA-mRNA codon couplings are ribosomes

- a ribosome is made up of a small and large subunit, each a group of proteins and (60% by mass) rRNA

- ribosomes conduct specific coupling of tRNA anticodons with mRNA codons during protein synthesis

- prokaryotic and eukaryotic ribosomes are similar but not the same

- each ribosome has a binding site for mRNA and 3 binding sites for tRNA on the large subunit (E,P,A)

1. Chain Initiation

- ribosome splits into its 2 subunits

- small subunit binds to mRNA and a special initiator tRNA (Met) with the help of initiation factors and GTP

- now large subunit binds

- initiator tRNA sits in the P site and A site is vacant

2. Chain Elongation - AA are added one by one to the growing polypeptide with

help from proteins called elongation factors i) codon recognition - mRNA codon in A site forms H-bonds with anticodon of

incoming tRNA carrying its appropriate AA - needs GTP hydrolysis for energy ii) peptide bond formation - peptide bond formed between polypeptide in P site and

newly arrived AA in A site - polypeptide separates from tRNA in P site and moves to

tRNA in A site

iii) translocation

- tRNA in P site dissociates from ribosome and is recycled

- ribosome moves along the mRNA and the tRNA in the A site carrying the growing polypeptide is shifted to the P site

- energy provided through GTP hydrolysis

• note : mRNA moved through ribosome in 5’ 3’ direction only

3. Chain Termination - elongation continues until a termination codon

reaches the ribosome’s A site (UAA, UAG, or UGA) - there are no anticodons for termination codons

because a release factor protein (not a tRNA) binds directly to the termination codon

- release factor causes ribosome to add water instead of an AA to the polypeptide

- polypeptide is released, tRNA leaves, ribosome separates

- with polyribosomes, several ribosomes can translate at the same time to make many copies

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POST-TRANSLATIONAL MODIFICATION

- chop Met? – probably

- certain AA may be modified with sugars, lipids, or P

- polypeptide may be cleaved

- 2 or more polypeptides may join (4o structure)