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5/7/08 Translation Additional reading assignment: Text pages 502-506 on protein sorting and In Sex Reversal, Protein Deterred by Nuclear Barrier http://fire.biol.wwu.edu/trent/trent/sexreversal.pdf animations of translation http://www.biostudio.com/demo_freeman_protein_synthesis.htm http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/translation.swf ultra-cool translation movie courtesy of Clint Spiegel (Chem dept) http://www.chem.wwu.edu/dept/facstaff/spiegel/steitz_movie_music_hi.mov

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From working copy (mRNA or transcript) to functional protein

How does this translation from one chemical language to another work? What’s in the translation toolkit

Shorthand abbreviation of part of the mRNA sequence of the SRY gene >gi|17488858|ref|XM_010627.4| Homo sapiens SRY (sex determining region Y chromosome) GGCAUGUGAGCGGGAAGCCUAGGCUGCCAGCCGCGAGGACCGCACGGAGGAGGAGCAGGAGCGCGGAGCCGCGAGCCCCGAGCCCCGAGCCCGGCGCCUGGCUGAGUAGAUGUCCAUGAGGAGCCCCAUCUCUGCCCAGCUGGCCCUGGAUGGCGUUGGCACCAUGGUGAACUGCACCAUCAAGUCAGAGGAGAAGAAAGAGCCUUGCCACGAGGCCCCCCAGGGCUCAGCCACUGCCGCUGAACCUCAGCCUGGAGACCCAGCCCGGGCCUCCCAGGAUAGUGCUGACCCCCAAGCUCCAGCCCAGGGGAAUUUCAGGGGCUCCUGGGACUGUAGCUCUCCAGAGGGUAAUGGGUCCCCAGAACCCAAGAGACCAGGAGUGUCGGAGGCUGCCUCUGGAAGCCAGGAGAAGCUGGACUUCAACCGAAAUUUGAAAGAAGUGGUGCCAGCCAUAGAGAAGCUGUUGUCCAGUGACUGGAAGGAGAGGUUUCUAGGAAGGAACUCUAUGGAAGCCAAAGAUGUCAAAGGGACCCAAGAGAGCCUAGCAGAGAAGGAGCUCCAGCUUCUGGUCAUGAUUCACCAGCUGUCCACCCUGCGGGACCAGCUCCUGACAGCC………………………….. etc, etc, etc, Shorthand abbreviation of the protein sequence coded for by the SRY gene >gi|17384045|emb|CAD13147.1| sex determining region Y [Homo sapiens] MQSYASAMLSVFNSDDYSPAVQENIPALRRSSSFLCTESCNSKYQCETGENSKGNVQDGVKRPMNAFIVWSRDQRRKMALENPRMRNSEISKQLGYQWKMLTEAEKWPFFQEAQKLQAMHREKYPNYKYRPRRKAKMLPKNCSLLPADPASVLCSEVQLDNRLYRDDCTKATHSRMEHQLGHLPPINAASSPQQRDRY

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How does the translation process work? How does this chemical conversion work? After the discovery of mRNA and the elucidation of the genetic code, molecular biologists puzzled over how a particular triplet of nucleotides could chemically code for a particular amino acid: In other words, how does a UGG codon ensure that the amino acid tryptophan is inserted into a polypeptide at a particular position in the polymer?

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Some scientists suggested that an amino acid R group interacted directly with its corresponding codon (triplet) via a “pocket” formed on the template that was complementary in shape and charge (or could form complementary H-bonds) to the amino acid side group

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BUT this idea was difficult to reconcile with the chemisty of nucleic acids and some of the R groups of amino acids Recall the various type of R groups found in amino acids: For which amino acids does this model make sense? For which amino acids does this model not make much sense?

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Francis Crick and the adaptor hypothesis Crick proposed a solution to this problem by envisioning an adapter molecule attached to each amino acid that would interact directly via hydrogen bonding with a triplet codon animation of translation showing adaptor http://www.blc.arizona.edu/INTERACTIVE/DNA3/protsyn95.mov

In essence Crick proposed the existence of a chemical go-between that was attached to an amino acid during protein synthesis and interacted via Hydrogen bonding with the codon on the mRNA

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Francis Cricks hypothetical adaptor molecule (bridging the chemical gap between RNA and protein) turned out to be the tRNA (transfer RNA) molecule tRNA is the chemical go-between that allows amino acids to “interact” with an mRNA template tRNA's as adaptors: Each amino acid has at least one tRNA Each tRNA reads the codon or codons specific for one amino acid For example tRNAser has an anticodon that reads only serine codons Before it participates in protein synthesis, a tRNA is “charged” with its corresponding amino acid

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REMEMBER that the CODE table is always presented in mRNA language CODON: mRNA language ANTI-CODON tRNA language LIKE OTHER H-BONDING INTERACTIONS BETWEEN NUCLEIC ACID POLYMERS, THE mRNA AND tRNA H-BOND IN AN ANTIPARALLEL FASHION

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Transfer RNAs are the chemical interpreters in translation: see also Figure 7-26 in text • A “charged” tRNA is a nucleic acid on one end:

capable of forming hydrogen bonds with an mRNA molecule

• It is an amino acid on the other end: capable of being incorporated into a polypeptide

A charged tRNA acts as a chemical interpreter because it speaks in both chemical languages. This tRNA is charged with the amino acid cysteine. Via its AGC anti-codon, it can base-pair with the cysteine codon UGC in an mRNA

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What does a tRNA look like? • Transfer RNAs are short RNA polymers ranging from

75- 85 nucleotides in length • Despite their small size, tRNAs have a fairly elaborate

secondary and tertiary structure In contrast mRNAs have little secondary structure

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Secondary structure • involves complementary base-pairing between different

portions of the molecule to form stem-loop structures held together by short stretches of intra-strand h-bonds

• All tRNAs have the same overall 2o and 3o structure but they differ in the sequence of the anticodon

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Tertiary structure • similar to proteins, the tertiary structure is produced by

folding the tRNA to produce a 3-D molecule with a characteristic L shape

• at one end of the L is a loop of polymer which contains 3 bases called the anticodon

• the anticodon hydrogen bonds with the corresponding codon on the mRNA

• at the other end of the molecule is an attachment site for the amino acid

Like proteins the tertiary structure of tRNAs is critical for their role in protein synthesis In contrast to proteins, all tRNAs fold up into the same shape NOTE: Genes that specify tRNA’s do not have a protein product: their final product is an RNA molecule

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AGT

TCA

TCA

UCA5' 3'

5'

3'

3'

5'

5' 3'

transcription

AGT

splicing and processing

in eukaryotes

DNA

UCAAGU

mRNA

serine codon

on mRNA

serine anticodon

on tRNA

5'

serine serine attached to

tRNA at 3' endser

3' 5'

Chemical conversion of TCA into serine.

Accuracy of translation depends on precise matching:

(1) of an amino acid with its cognate tRNA

(2) of the anitcodon of a charged tRNA with its corresponding

codon on the mRNA

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Peptide bond formation always takes place on the surface of complex structures called ribosomes The ribosome orchestrates peptide bond formation:

1. It binds the charged tRNA and mRNA in the proper spatial orientation

2. This binding facilitates accurate decoding of the mRNA and the rapid formation of the peptide bond

3. It contains the polypeptide polymerase (real name is peptidyl transferase) which catalyzes peptide bond formation

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The two essential functions of the ribosome: • The small subunit contains the messenger RNA decoding site

• the large subunit the peptidyl transferase centre.

• The mRNA threads through the subunit interface, and is decoded.

• The three transfer RNA binding sites, A (acceptor), P (peptidyl) and E (exit) then handle amino-acid selection, addition and completion of polypeptide synthesis. Here a charged tRNA is shown in the A site, and a nascent peptidyl-tRNA in the P site; the E site is vacant.

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Essential cell biology: animation of translation 07.6 The ribosome catalyzes formation of the peptide bond The catalytic function responsible for peptide bond formation is called peptidyl transferase The incorporation of an amino acid into a growing polypeptide chain. the polypeptide grows by stepwise addition of amino acids to its C-terminus. The figure shows the addition of the 4th amino acid to the chain.

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The ribosome • is a particle that is found in abundance in

all cells that are actively making protein • most complex component of the protein

sythesizing machinery of the cell • made of ribosomal RNA (called rRNA)

and a large number of proteins • rRNA has elaborate tertiary structure

formed by intrastrand hydrogen bonds

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Ribosomes in the cytoplasm of a eukaryotic cell. This electron micrograph shows a thin section of cytoplasm. The ribosomes appear as black dots (red arrows). Some are free in the cytosol. Others are attached to the endoplasmic reticulum

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• Rough ER has ribosomes attached to the membrane

surface

• these ribosomes produce proteins that will be secreted

by the cell (such as insulin in the pancreas) as well as proteins that are imbedded in the plasma membrane

Essential cell biology: animation of polyribosomes 07.7

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A comparison of the structure of eukaryotic and prokarytoic ribosomes. Despite the differences in size (indicated by the S value) both prokaryotic and eukaryotic ribosomes have nearly the same structure and they function similarly

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Elongation of Polypeptides during translation See also figures in text – 7-31 NOTE: A, P and E sites NOTE; energy source is GTP, a relative of ATP Be sure to look at these animations http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/translation.swf http://fire.biol.wwu.edu/trent/trent/06.7-translation_II.mov OPTIONAL: See also this animation showing the clever mechanism by which bacterial cells align mRNAs with the “starter” AUG (setting the reading frame) http://www.biostudio.com/demo_freeman_protein_synthesis.htm

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Science 285: 2077 9/24/99 Rotund marvels. Structure of the 70S ribosome and its functional center. (Top) The tRNA molecules span the space between the two subunits; the channel in the 50S subunit through which the growing peptide chain protrudes is shown in dashed lines. (Bottom) The 30S (left) and 50S (right) subunits have been opened up to give a better view of the three binding sites for tRNA, the A, P, and E sites. The 30S subunit shows the approximate location of the site where the codons of the mRNA are read by the anticodons of the tRNAs. The 50S subunit has the tRNA sites shown from the opposite direction. The acceptor ends of the A- and P-site tRNAs are close to each other in the peptidyl transfer site, which is close to the exit channel located behind a ridge in the 50S subunit. The binding site for EF-G and EF-Tu is located on the right-hand protuberance of the 50S subunit.

50S = large subunit 30s = small subunit (for prokaryotes)

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1/28/02 Ribosomes and ribozymes

prokaryotic ribosome -- 3 bound tRNAs rRNAs: cyan (16S), gray (23S) and light purple (5S) A, P, E site tRNAs in yellow, orange, red (respectively) small subunit proteins: dark purple large subunit proteins: magenta rRNA - ribosomal RNA tRNA = transfer RNA\

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All ribosomes contain a large and small subunit. Each subunit is made of various polypeptides and rRNA (ribosomal RNA)

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For many years, the rRNA component of the ribosome was viewed as an inert structural matrix for the proteins that carried out the catalytic business of the ribosome Conventional wisdom said all biological catalysts are made of protein So biochemists spent many years looking for the catalytic activity among the various ribosomal proteins But they ran into a problem: they couldn’t seem to assign this function to a particular protein Discovery that RNAs could act as catalysts in an unrealted process set a precedent for thinking about rRNA as the business end of the ribosome -- the catalytic componant The discovery of RNA catalysis changed the way many scientists viewed the role of RNA in the ribosome Perhaps the proteins in the ribosome provide an inert structural matrix for catalytic RNA ribozyme: an RNA molecule that functions as a catalyst

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MAKING THE PEPTIDE BOND Figure 2 Interactions of the CCA ends of ribosome-bound tRNA with the large ribosomal subunit. a, Cut-away view of the large ribosomal subunit with tRNAs bound. tRNAs are positioned on the large ribosomal subunit as described in the legend for Fig. 1, and the subunit sliced in half along a plane approximately perpendicular to the Fig. 1 plane of view to reveal the placement of the acceptor stems of ribosome-bound tRNAs in the large subunit's peptide exit tunnel. rRNA is shown in white, and ribosomal protein in yellow. tRNAs are colour coded as follows: E site, brown; P site, purple; and A site, green. b, Interactions of the CCA sequences bound in the P site and the A site with 23S rRNA. The molecule bound in the P site is the deacylated tRNA analogue CCA (purple). The molecule bound in the A site is the peptidyl-tRNA analogue CCA–puromycin– phenylalanine–caproic acid–biotin (green)15. Blue bases are components of the P loop29; brown bases belong to the A loop30. The nucleotides in cyan and red are other components of the peptidyl transferase centre. Bases are numbered according to the sequence of H. marismortui 23S rRNA. The two 23S rRNA bases closest to the newly formed peptide bond are A2486 and U2620, which correspond to A2451 and U2585 in E. coli, respectively. (Reproduced with permission from ref. 15.)

cutaway of large ribosomal subunit i yellow = protein white = rRNA rRNA: red and cyan in active site tRNAs: brown = E site purple = P site green = A site active site: peptide bonds are formed between amino acids attached to the tRNAs in the A and P sites(catalyzed by rRNA in large subunit) Exit tunnel: polypeptide is fed through this tunnel in the ribosome

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ultra-cool translation movie courtesy of Clint Spiegel (Chem dept) http://www.chem.wwu.edu/dept/facstaff/spiegel/steitz_movie_music_hi.mov

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Nucleic acids are not obvious candidates for being biological catalysts Since nucleic acids are not as chemically diverse as proteins, RNA-based catalysis is not as powerful or vesatile as protein-based catalysis: • Ribosomes synthesize polypeptides at a rate of about

20 amino acids per second. • Compare this speed with that of DNA polymerase, a

protein-based catalyst, which polymerizes >500 nucleotides per second.

• On the other hand, translation is a pretty accurate process: only one in every 10,000 codons in mRNA is decoded incorrectly.

Such RNA catalysis is probably a vestige of our evolutionary past: During the very early history of life on earth, RNA may have played the roles that DNA and protein play today; • the chemical reservoir of genetic information • the biological catalysts Click below to explore this topic in more detail http://fire.biol.wwu.edu/trent/trent/ribosomeevolution.pdf

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Science 19 December 2003:Vol. 302. no. 5653, p. 2050DOI: 10.1126/science.302.5653.2050a NEWS OF THE WEEK CELL BIOLOGY: In Sex Reversal, Protein Deterred by Nuclear Barrier Aparna Sreenivasan* SAN FRANCISCO, CALIFORNIA--Each year, a few babies are born with a male set of chromosomes and female sexual organs. This sex reversal, called Swyer syndrome, can happen when changes in a protein called SRY impair its function. Previously reported SRY mutations interfere with the protein's ability to bind to DNA. But recent data, including some presented here this week at the annual meeting of the American Society for Cell Biology (ASCB), show that in some cases, the altered protein has trouble entering the nucleus of fetal male gonadal cells. Thus, genes that should be turned on by SRY to make testes remain off. "We now show a completely different mechanism as to how someone can become an XY female," reports cell biologist David Jans of Monash University in Victoria, Australia.

Getting certain proteins in and out of the nucleus is important for normal cellular functions. But Jans, Vincent Harley of Prince Henry's Institute of Medical Research in Victoria, Australia, and colleagues were the first to directly link a defect in nuclear import with a human syndrome. They reported this summer that SRY molecules engineered to have the same changes found in some sex-reversed people seemed to have problems getting into the nucleus of cells. Normal SRY slips into the nucleus readily.