Topic 13: Protein Synthesis

auxotrophs

nutritional mutants

one-gene-one enzyme hypothesis was updated to the…

one gene-one polypeptide hypothesis

one gene-one polypeptide hypothesis

each gene codes for an animo acid that forms a polypeptide chain

central dogma 

DNA→RNA→proteins 

protein coding gene

a gene that codes for a protein, transcribed into mRNA

Are all genes protein coding genes?

no, some encode various molecules that function in transcription, translation, and other processes in the cell

Where does transcription occur in eukaryotes? What does it produce?

nucleus, precursor (pre) mRNA that must be altered into functional mRNA

In eukaryotes, where does functional mRNA get translated?

the cytoplasm

Where does transcription occur in prokaryotes and what does it produce?

cytoplasm, produces functional mRNA (no processing/modifications needed) 

genetic code

the nucleotide info that specifies the amino acid sequence of a polypeptide

codon

each three letter word (triplet) of the genetic code

How many codons do the 4 mRNA nucleotides make?

64

61 of the codons are…

sense codons which specify amino acids

one codon is the…

start/initiator codon

start/initiator codon

AUG, codes for methionine and is always the first codon read in translation

3 of the codons are…

stop codons 

stop codons

UAA, UAG, UGA, don’t code for an amino acid and stop polypeptide synthesis

What are the only two amino acids that are coded for by a single codon?

methionine and tryptophan 

degeneracy

describing an amino acid that is coded for by multiple different codons

What does it mean to say that the genetic code is commaless?

there are no indicators to mark the end of codon and the beginning of the next

What does it mean to say that the genetic code is universal?

it’s essentially the same in all living organisms and viruses

What does a gene in eukaryotes consist of?

a promotor (with a TATA box) and transcriptional unit

What are the three stages of transcription?

1. initiation

2. elongation 

3. termination 

initiation 

molecular machinery assembles at the promoter and begins synthesizing an RNA copy of the gene 

In transcription, what direction is RNA made, what direction is DNA read?

RNA is made in the 5’→3’ direction, DNA is read in the 3’→5’ direction 

elongation 

• RNA polymerase II moves along the gene, extending the mRNA chain

• DNA continues to unwind ahead of the enzyme 

termination

a specific termination sequence is reach and the RNA transcript and RNA polymerase II are released from the DNA template

What do prokaryotes have and have not in transcription that eukaryotes dont?

• have: terminators - specific DNA sequences that end the transcription of a gene

• don’t need transcription factors, bind RNA polymerase binds directly to DNA 

What do eukaryotes have and have not in transcription that prokaryotes dont?

• have: transcription factors that help RNA polymerase II bind to the template strand

• don’t have: terminators 

noncoding RNA genes

genes encoding RNA that aren’t translated

RNA polymerase III

responsible for transcribing the genes for tRNA and one of the 4 rRNAs

RNA polymerase I

responsible for transcribing the genes for the other three rRNAs

Do prokaryotes have multiple RNA polymerases?

no

How are the promoters for specialized noncoding RNA genes?

specialized for the correct RNA polymerase type

What are the two bookends of eukaryotic pre-mRNA?

• 5’ untranslated region (5’ UTR)

• 3’ untranslated region (3’ UTR)

What is the 5’ end of pre-mRNA?

5’ methyl cap

5’ methyl cap

• consists of a guanine-containing nucleotide that is revered so that its 3’ OH group faces the beginning of the molecule

• connected by 3 phosphate groups 

• adding by capping enzyme

• the site where ribosomes attach to mRNAs at the start of translation 

What does polyadenylation signal and poly (A) polymerase) add to the 3’ end of a pre-mRNA?

poly (A) tail 

poly (A) tail

• 5-240 adenine nucleotides 

• protects the pre-mRNA from attack by RNA digesting enzymes in the cytoplasm

mRNA splicing

occurs in the nucleus and removes introns from the pre-mRNA and joins exons together

Where does splicing take place?

in a spliceosome formed from between pre-mRNA and several small ribonucleoprotein particles (snRNPs) which are made up of small nuclear RNA (snRNAs) that are bound to a bunch of small proteins

lariat structure

the released intron from splicing, called this bc of its shape

alternative splicing

reactions that join exons in different combos to make different mRNAs and proteins from a single gene

What is a pro of alternative splicing?

it greatly increases the number and variety of proteins encoded in the cell nucleus without increasing the size of the genome

What is an example of alternative splicing?

the a-tropomyosin gene is alternatively spliced into: 

• smooth muscle 

• skeletal muscle 

• fibroblast

• liver

• brain 

exon shuffling 

a process by which existing proteins regions or domains are mixed into novel combos, produces changes more efficiently and quicker than by changes in individual amino acids at random points 

What did ENCODE’s study of transcriptomics reveal?

pervasive transcription of the genome and variable expressions of gene isoforms 

gene isoforms

protein-coding genes that are transcribed to different forms of mRNA

In what direction is mRNA read in translation?

5’→3’ direction

In what direction is a polypeptide assembled in translation?

N-terminal end → C-terminal end

What shape does tRNA wind into?

four double-helical segments that create a cloverleaf pattern

anticodon

3 nucleotide segment that base pairs with codons in mRNAS

pairing of the anticodon with which nucleotide is always precise?

the first 2

Which nucleotide in the codon does the anticodon have more flexibility pairing with?

the third one 

the same tRNA anticodon can read codons that have either….

the C or U in the third position

What does the special purine isosine allow tRNA

more wobble, allowing it to pair with codons that have either U, C, A in the 3rd position

aminoacylation/charging 

the addition of the correct amino acid to tRNA, producing an aminoacyl-tRNA, requires ATP

What enzymes facilitates aminoacylation and how many are there?

aminoacyl-tRNA synthetases, there are 20, one for each amino acid

ribosomes

ribonucleoprotein particles that translate mRNA into chains of amino acids

Where are ribosomes located in eukaryotes?

they’re either suspended in the cytoplasm or attached to the rough endoplasmic reticulum (RER)

What are ribosomes composed of?

ribosomal RNA (rRNA) and ribosomal proteins

• a large subunit 

• a small subunit 

What are the three bonding sites that tRNAs interact with the ribosome

1. aminoacyl site (A site) 

2. peptidyl site (P site) 

3. exit site (E site)

aminoacyl site (A site)

aminoacyl-tRNA carrying the next amino acid to be added to the polypeptide binds here

peptidyl site (P site)

tRNA carrying the growing polypeptide chain is bound here

exit site (E site)

tRNA without an amino acid bind here before exiting the ribosome

What are the three stages of translation?

1. initiation

2. elongation 

3. termination 

initiation (translation)

components assemble on the start codon (AUG)

elongation (translation)

assembled complex reads the string of codons in the mRNA 1 at a time while joining specified amino acids into a peptide 

termination (translation)

complex disassembles after the last amino acid specified by mRNA has been added 

Translation initiation in eukaryotes is aided by?

initiation factors (IFs)

What do prokaryotes use for translational initiation instead?

directed by a ribosome binding site

What protein faciliates an aminoacyl-tRNA binding to a codon in the A site?

elongation factors (EFs)

peptidyl transferase 

catalyzes polypeptide bonds between the C-terminal end of one amino acid, and the N-terminal of the next

release factor (RF)/ termination factor 

tRNA, the polypeptide chain, and the mRNA chain are released from the ribosome 

polysome 

multiple translation sites on each mRNA, the shorter the peptide sequence, the more recent the attachment, with short peptides being closer to the 5’ end, and the longer peptides being closer to the 3’ end

What are the three compartments that proteins can be sorted to?

1. cytosol 

2. endomembrane system

3. other organelles  

protein sorting; cytosol

• synthesized on free ribosomes

• released into the cytosol 

• get folded into secondary, tertiary, and quarternary structure to state functioning as proteins 

protein sorting: endomembrane system

• begin synthesis on free ribosomes 

• ribosomes directed to ER or Golgi complex

• peptides are released into ER or the Golgi complex 

• undergo further packaging, folding, or marking 

• ribosomes constantly binding/detaching from RER 

protein sorting: other organelles

• synthesized on free ribosomes 

• send to mitochondria, chloroplast, nucleus