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Transcribing rRNA genes

•  Produces three rRNAs
•  Assembly requires a very large number of non ribosomal components

Ribosomal RNA gene is repeated, when transcription begins, multiple mrnas all at the same time. ribosomes made quickly. want plenty of ribosomes available to make proteins. Ribosome is many proteins

RNA Pol I translates 3 subunits, 5S subunit by RNA Pol III

1

What is a ribosome composed of?

Ribosome is composed of many proteins and ribosomal RNA. Ribosomal RNA can form RNA RNA hybrids between mRNAs and tRNAs during initiation of translation

2

Ribosome Biogenesis

Ribosomes made in nucleus. Certain proteins associated with it, then some of those proteins will move away. Ribosomes move closer to the cytoplasm, as they move through the nucleolus they are associated with different proteins, then move out to the cytoplasm and associated with more proteins.

3

Svedberg units

Refers to sedimentation, how quickly it's spun to the bottom of tube- how heavy it is

4

Are ribosomes enzymes?

Yes, ribosome has enzymatic activity, region that catalyzes reaction for peptide bond formation in between amino acids. Spliceosome is also considered a ribozyme, has RNA and makes RNA RNA hybrids.

5

Membrane bound ribosomes

Produce proteins that will be used in the plasma membrane or will be secreted from the cell

Typically membrane bound. rough ER membrane is continuous with the nuclear membrane, ribosomes give it studded appearance. Proteins translated on rough er usually have transmembrane regions, will be transported out to the cellular membrane, or they will be secreted from the cell. Things usually modified in rough er, lipids added in smooth er, Golgi collects up similar proteins and anchors them to a secretory vesicle and then exported to the cellular membrane

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Cytosolic ribosomes

–  Produce proteins to be used in the cytosol
–  Highly reducing environment in side the cell

Cytosolic proteins are going to be used in the cell. No disulfide because of the reducing environment

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Ribosomes and signal peptides

Mrnas meant to be translated in the roughER have signal peptide.

Once they bind to a ribosome it starts the amino acids, and the first few AA in the peptide will be a signal peptide sent to signal recognition particle, which binds to it. Conformational recognition.

Then binds to a receptor in rough ER, protein made, translated, and fed into the lumen of the rough ER. Proteolytic processing removes the signal peptide. Once small subunit disassociates, can go back to the cytosol and translate more

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How does an mRNA triplet specify an amino acid?

•  How would a nucleicacid base interact with an amino acid incapable of forming H bonds?

tRNA

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Experiment: are amino acids that are attached to tRNA molecules transferred to proteins?

Radioactive leucine attached

Add aminoacyl tRNA to in vitro translation system, included mRNA(something to be read) and ribosomes (something to read)

Radioactive amino acids found in proteins

Conclusion: aminoacyl tRNAs transfer amino acids to growing polypeptide chains

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tRNA + aminoacid

•  A tRNA covalently linked to an aminoacid is called an aminoacyl tRNA

Linked through enzyme called aminoacylsynthetase.

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Accidental Adapter Finding

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Accidental Adapter Finding

•  Researchers working in a cell free protein synthesis system found that a few things were needed: ribosomes, mRNA, amino acids, ATP, and GTP

•  It wouldn’t work unless they included a cellular fraction - without this fraction protein synthesis would not occur (in the fraction was tRNAs

13

How is the secondary structure of RNA made?

Stems are created by hydrogen bonding between complimentary base pairs

Loops consist of unpaired bases

14

What do modified bases do?

Modified bases important in tRNA recognition by aminoacyl trna synthetase

15

The structure of a tRNA

All tRNAs about 70-80 base pairs

Pots transcriptional modifications to bases in tRNA

Complimentary regions

The loop at the base is always formed of seven nucleotides, the middle three are the anticodon

Acceptor arm and variable region

16

Acceptor arm

3' end, free OH where the AA will be bound to the tRNA

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Variable arm

Variable arm important for recognition by aminoacyl tRNA synthetase

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Anticodon

3 positions vary in which bases are there, but recognizes codon on mRNA and dictates which amino acid should be incorporated into the growing polypeptide

19

tRNA tertiary structure

L shaped

(One end attaches AA, one is the anti codon)

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Activation of tRNA’s

•  Each tRNA must be attachedto the proper amino acid (If tRNA is carrying amino acid, we call it charged, or activated, loaded)

•  Process is mediated by aminoacyl-tRNA-synthetase

•  Recognition of tRNA by aminoacyl-tRNA-synthetase
–  Anticodon is of major importance
–  Enzyme has a pocket for the anticodon
–  There are also other sites that help with recognition (length of variable arm, modified bases)

21

Reactions of tRNA with the enzyme

Active site on aminoacyl tRNA synthetase binds ATP and amino acid by an ester bond (enzyme specific to one amino acid)

Reaction leaves AMP and amino acid bound to enzyme and two phosphate groups are released. "Activated" amino acid has high potential energy

Activated amino acid transferred from tRNA synthetase to the tRNA specific to the amino acid, resulting in aminoacyl tRNA which is ready for translation

22

Where exactly is the amino acid added on the tRNA?

Depending which oh aa is added to, class two or class one. Class two goes onto 3' carbon, class one goes onto the 2' carbon

23

The Genetic Code

•  Have an mRNA that is carrying a message, that message has to be decoded

•  The “code” for amino acids are nucleotide triplets

•  The code is non-overlapping (3 adjacent codons)

•  Have 4^3 = 64 possible combinations to code for amino acids, but there are only 20 amino acids

Conclusion: The DNA code is degenerate

24

Wobble at the the third position

•  One tRNA can recognize more than one codon - wobble at the third position. There's only 3bp so there's nothing to recognize change in geometry. Not like continuous DNA strand. U/G base pair is not a bad match

•  Two codons that specify the same amino acid and only differ at the third position, should use the same tRNA in protein synthesis

tRNA matched up opposite to mRNA (3' to 5')

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What is base "I"?

I is deaminated adenine. Only found on tRNAs, perfectly good. Base analog, binds with U, C or A

26

Translation Occurs in Three steps

Initiation - finding the start codon and assembling the ribosomal subunits

Elongation - reading the mRNA sequence and polymerizing the corresponding amino acids

Termination - recognition of the stop codonand release of the polypeptide

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Ribosome subunits in prokaryotes

Each subunit is composed of a set of proteins complexed with one or more ribosomal RNAs.

The large subunit also has an accessory RNA

The large rRNA molecule in each ribosome sequentially binds the aminoacylated ends of the incoming tRNAs and catalyzes peptide bond formation

Together the subunits are 70s

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Small subunit in prokaryotes

16s RNA + proteins = 30s

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Large subunit in prokaryotes

23s RNA + 5s RNA + proteins = 50s

5 and 23 transcribed by different polymerases

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Prokaryotic initiation

•  Small ribosomal unit will bind to the start codon AUG, tRNAfMet comes in after

•  Shine-Dalgarno sequence recognized

•  Initiation Factors
–  IF1
–  IF2
–  IF3

Uac anticodon binds. If3 and if1 leave, ribosomal assembles

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Prokaryotic start codon

N-formylmthionine

tRNA bound to methionine must enter that site

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Shine-delgarno sequence

resides 5-10 nucleotides before the start codon, recognized by 16S of the small subunit

In mRNA there is an aggagg, compliment to ribosomal RNA in small subunit. This is a recognition sequence that dictates where the ribosome will sit on mRNA.

33

IF1 and IF3

One and three keep the small and large ribosomal unit separated. Channels that go through the whole ribosome and in order for them to make the correct conformation they have to assemble on the mRNA a specific order. If the small and large were bound they couldn't recognize the sequence

34

IF2

If2 bound to gtp sit down on the codon and brings in the first tRNA. tRNA is bound to n-formylmethionine.

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Difference in eukaryotic translation

•  Transcription and translation are coupled in prokaryotes (no introns, as long as there is enough mRNA out of pol II the ribosome will assemble)

•  A eukaryotic mRNA is already fully processed- Not coupled, mRNA has to be transported out of the nuclear pores and into the cytoplasm. When in cytoplasm then bound to ribosome to be translated
–  May have folded into secondary or tertiary conformations
–  Coated in RNA binding proteins

The 5’ cap and polyA tail play an important role in translation

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Cap dependent

Initiation proteins recognize the mRNA 5'-5' cap (eIF4E)

Protein factors bind the small ribosomal subunit (40s)

Poly A binding protein recognizes the mRNA tail (PABP)

eIF4A scans for start codon (AUG)

37

Cap independent

Secondary structures in mRNA that are recognized by initiation factors

IRES (site of secondary structure), allows translation to begin in the middle of the mRNA

eLF4A scans for start codon

PABP recognizes poly A tail

38

Other things about eukaryotic translation

40s subunit with eIF3 and eIF1A, eLF2-GTP, and the tRNA make up the 43s complex

PABP, eIF4G, eIF4A the mRNA and eIF4G (cap dependent only) make up mRNA complex

•  eIF3 and eIF4G bring the two together, keep subunits from binding too early
•  Once the 43S complex has bound the mRNA it willscan for the appropriate AUG start codon
•  In eukaryotes the Kozak sequence (5’-CCACCAUGC – 3’) is a conserved sequence
containing the start codon

39

tRNA Binding Sites

There is always a free codon to bind to the tRNA

•  A Site - holds the Aminoacyl tRNA (already been charged)

•  P Site - holds the Peptidyl tRNA. Also first charged trna with methionine with tRNA has to be there because p site has peptidyl transferase activity. Catalyzes formation of peptide bond, which goes from AA in p site to AA on tRNA in a site. AA attatched to tRNA in p site will be transferred through peptide bond to AA joined to tRNA in a site

•  E Site - holds exiting uncharged tRNA after release from mRNA

40

Elongation factors

•  EFTu - brings in each new Aminoacyl tRNA to the A site. Only h bonds keep appropriate tRNA in the A site. Then peptidyl transferase activity can happen

•  EFG - drives the translocation step after peptidyl transfer. Peptidyl transferase activity and then peptide bond formed between the two amino acids. Now AA in p site is uncharged and joined to AA in a site. Shift down one codon, methionine trna is in e site, and it exits. Now Dipeptide in p site. Another charged tRNA comes into the a site.

( Both hydrolyze GTP)

•  EFTu is recycled by EFTs

41

What happens to the deacetylated tRNA when you release it?

Leaves to interact with enzyme again

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Prokaryotic termination

• RF1 recognizes UAG and UAA. RF2 recognizes UGA and UAA. (Remember the codon table)

• Both require the GTPase RF3 (enhances the other two releasing factors).

43

What happens after the binding of release factors?

• After binding of RF1 or 2 with RF3-GTP into the A site
1. GTP is hydrolyzed, the subunits are separated(no more peptidyl transferase activity)
2. the new polypeptide is cleaved from the tRNA.

• The large and small subunit are released and all of these components can then be recycled to begin translation of the next polypeptide.

44

When does translation begin in bacteria?

In bacteria, ribosomes begin translating mRNA while the mRNA is being synthesized

45

When does translation begin in eukaryotes?

Intron splicing occurring at the same time that mRNA is being made, then processed and translated.

46

How many ribosomes can translate one mRNA?

Several ribosomes can translate one mRNA simultaneously

47

What is a protein's orientation with the mRNA?

•  The 5’ to 3’ polarity of the mRNA corresponds to the N to C polarity of the protein.

•  Ribosomes translate themRNA from 5’ to 3’, synthesizing the protein from the N-terminus to the C-terminus

• Polypeptides grow by addition of amino acids one at a time to the C-terminal end of the growing polypeptide chain.

• Polypeptides are synthesized N-terminus to C-terminus

48

What is the direction of replication, transcription, and translation?

• DNA synthesis is 5’ to 3’

• RNA synthesis is 5’ to 3’

• Ribosomes move on mRNA in the 5’ to 3’ direction.

49

What roles does RNA play in protein synthesis?

•  16s rRNA attracts mRNA through RNA-RNA interaction. Need to locate shine dalgarno, complimentary rRNA and mRNA. They base pair and small subunit stays there, then aug is start codon

•  tRNA makes direct contacts with 16s and 23srRNAs.

•  peptidyl transfer reaction is catalyzed by 23s rRNA. Peptide synthesis is RNA-mediated.

50

What could go wrong?

If we change the reading frame, there are mutations

Problems occur in writing the message or reading the code