BIO2089 Translation

Translation =

= protein synthesis

In bacteria transcription & translation

both occur in the cytoplasm & are linked

In eukaryotes transcription & translation

occur separately, transcription in the nucleus & translation in the cytoplasm

Nucleobases of DNA (deoxyribonucleic acid)

T, C, A, G

Nucleobases of RNA (ribonucleic acid)

U, C, A, G

mRNA

Messenger RNA

tRNA

Transfer RNA

rRNA

ribosomal RNA

Cells contain ___ types of RNA

several

mRNA is the template for

translation - the cell reads the info in the mRNA to build a protein

Bases in mRNA

U, C, A, G

mRNA bases are read in groups of

3 nulceotide triplets called codons

Each codon represents

an amino acid

How many possible codons are there

4³ codons = 64

How many codons encode amino acids

61 / 64

How many codons encode for stop

3 / 64 = UAA, UAG, UGA

How many amino acids are there

20

Can amino acids have more than 1 codon

yes, there are 61 codons but 21 amino acids & amino acids that are found more commonly in proteins are represented more often than amino acids found more rarely eg Arg has 6 but Trp has 1

AUG (methionine) is usually the

start codon

mRNA is recognised by __ because

(adapter transfer RNA) tRNA, it cannot be recognised directly by an amino acid

Adapter tRNA contains

a trinucleotode anticodon that is complementary to the codon in the mRNA

tRNA is linked to a

specfific amino acid at the 3’ end

tRNA structure

single chain of ribonucleotides, consists of H-bonded stems & loops making the tertiary structure and L-shape / clover leaf

there are __ bases in tRNA

modified, >50 types known, some are methylated/dimethylated versions of A, U, C & G

Inosine is a

modified base found in the anticodon of tRNA

How many tRNAs are there per cell

~45

Can 1 tRNA recognise more than 1 mRNA codon

yes, there are 61 codons but 45 tRNA

What is degneracy

tRNAs can recognise more than 1 codon & the 1st 2 bases are the same but the 3rd one differs

Degeneracy is possible because of the

wobble hypothesis proposed in 1966 by Francis Crick

what is the wobble hypothesis

Normal base pairing occurs between bases 1 & 2 but the 3rd base is less spatially confined & pairing is less stringent so can wobble forming non-standard base pairing

In bacteria mRNA wobble codon base in postion 3 binds to which possible tRNA bases

• A = U or Inosine

• C = G or Inosine

• G = C or U

• U = A, G, Inosine

In eukaryotes mRNA wobble codon base in postion 3 binds to which possible tRNA bases

• A = U

• C = G or Inosine

• G = C

• U = A, G, Inosine

Charging tRNA with amino acids

• Carboxyl group of AA is attached to hydroxyl group at 3’ end of tRNA

• Catalysed by aminoacyl-tRNA synthease = AA & ATP bind to this enzyme then tRNA bonds

• Coupled to ATP hydrolysis

aminoacyl-tRNA syntheases are

specific for their correct amino acid

tRNA with linked AA is called

charged

tRNAs charged with the same AA are called

cognate tRNAs

In eukaryotes is there an aminoacyl-tRNA synthease for each amino acid

yes each AA has its own enzyme to link it to the right tRNA

In bacteria is there an aminoacyl-tRNA synthease for each amino acid

No, the same enzyme is sometimes responsible for coupling more than 1 AA & there’s a 2nd enzyme that modifies the incorrect AA

Most aminoacyl-tRNA syntheases select the correct AA by a

two-step mechanism that recognises specfific factors of both the AA and the tRNA

aminoacyl-tRNA synthease active site

the correct AA has the highest affinity for the active site, larger AA don’t fit the active site

aminoacyl-tRNA synthease editing site

the correct AA doesn’t fit in the editing site, it only accepts incorrectly linked AAs which are hydrolysed & removed = proff reading

Polypeptide chains in bacteria start with a

modified AA = N-formylmethionine (fMet)

fMet is carried by

a special initiator tRNA (tRNAf) that participates in initation

In eukaryotes the initator tRNA carries

methionine rather than fMet

Ribosomes are the site of

protein synethsis (translation)

Ribosomes are

huge catalytic machines (riboenzymes) containing rRNAs & ~50 proteins

Ribosmes are divided into

large & small subunits

Ribosome small subunit

tRNAs are matched to codons

Ribosome large subunit

links AA together by peptide bond catalysis

Different cellular components of ribosomes separate into

distinct bands when centrifuged through a gradient & the relative rate of sedimentation is dependent on the size & shape = described by sedmimentation coefficent (S, svedberg unit)

Bacteria ribosomes

70s (50S & 30S )

Eukaryote ribosomes

80S (60S & 40S)

When not actively synthesisng protein the large & small subunits of a ribosome are

seperate

Most of the RNA in a cell is rRNA which forms the

core of the ribosome & the rNA is essentail for its mechanism

A few bases in rRNA are

universally conserved - A1493 forms hydrogen bonds with the bases in the codon & anticodon if they are paired correctly

Steps of translation

Initiation , elongation, termination

Ribosomes have _ tRNA binding sites

3 = A (aminoacyl) , P (peptidyl) & E (exit)

The mRNA being translated is bound within the

small subunit of the ribosome

The tRNAs are in contact with both

large and small subunits of the ribosome

tRNA binding sites only form when

the 2 ribosome subunits come together

Polypeptide chain exits the ribosme via the

tunnel at the back of the P site

What is required for initation

Three protein initiation factors (IF1, IF2 and IF3)

30S forms a complex with

IF1 & IF3

IF2 binds

GTP and interacts with the initiator fMet-tRNAf

Does IF2 recognise tRNAs other than fMet-tRNAf

no

IF1 is bound near the A site to do what

direct fMet-tRNAf to the P site & subsequent amino acids enter at the A site

Positioning of the 30S subunit is controlled by the ___ (in bacteria)

Shine-Dalgarno (SD) sequence

The initiating AUG must be discriminated from internal AUG sequences, so is only 1 part of the initation signal, its also

preceded by the Shine-Dalgarno (SD) sequence, which is purine-rich

Shine-Dalgarno (SD) sequence is recognised by

the ribosome 16S rRNA (in the small subunit)

what leads to the ejection of IF1 and IF3

structural changes

IF2 stimulates the

association of the 50S subunit

What is bound to IF2 that allows its release

GTP, its hydrolysed & IF2 released

In eukaryotes Met-tRNAi (initatior tRNA) is loaded onto the small subunit with

eukaryotic initiation factors (eIFs)

In eukaryotic initation the small subunit recognises

5’ cap of an mRNA molecule

Eukaryotic initation requires

the poly-A tail, bound by poly-A binding proteins

In eukaryotes there is no

Shine delgrano sequence so uses 5’cap & poly A tail as signal to position the ribosome in the right place

In eukaryotes initation the small subunit

moves 5’ to 3’ along the mRNA searching for 1st AUG powered by ATP dependent helicases where the eIFs dissociate and the large subunit assembles

A eukaryotic mRNA generally encodes ___ protein

1

Bacterial mRNAs encode ___ proteins

several

why can Bacterial mRNAs encode several proteins

Bacterial ribosomes can assemble anywhere on an mRNA molecule with a ribosome binding site

Polycistronic

several proteins can be translated from the same mRNA molecule

Elongation

bringing in correct aminoacyl-tRNA, proof reading, peptide bond formation, mRNA translocation

elongation Requires proteins called

elongation factors to bring the aminoacyl tRNA into the ribosome

Aminoacyl-tRNA is delivered to the ribosome A site by

the elongation factor Tu (EF-Tu), a GTPase

• EF-Tu binds aminoacyl-tRNA in its GTP bound form

• EF-Tu interacts with the 16S rRNA

• Correct codon recognition causes a structural change in the 30S subunit that activates the GTP hydrolysis in EF-Tu

• The conformational change rotates the aminoacyl-tRNA in the A site so that it is brought closer to the aminoacyl-tRNA in the P site in a process called accommodation which aligns the AAs for peptide bond formation

• EF-Tu-GDP is released from the ribosome

• Released EF-Tu is reset into its GTP form by EF-Ts which bind to the GDP form - GDP dissociates & EF-Tu can be recharged with GTP

How is initator methionine prevented from being brought into an internal AUG sequence or into the A site (exclusively targeted to the P site)

EF-Tu does not bind fMet-tRNAf

The link between amino acids is a

peptide bond

Peptide bond formation is catalysed by

peptidyl transferase in the large subunit

Peptide bond formation is

thermodynamically spontaneous

The peptide chain is formed in the

A site; the subunits rotate and the peptidyl-tRNA is moved to the P site

mRNA translocation is catalysed by

elongation factor G (EF-G)

mRNA transolaction

• GTP is hydrolysed and mRNA is moved on by one codon

• The next codon is then positioned in the A site for the next aminoacyl-tRNA

• The deacetylated tRNA moves from the P site to the E site where it is released

• The peptide chain remains in the P site throughout continuous cycles, growing into the exit tunnel

Termination the end of translation is signalled by

one of three STOP codons – UAA, UAG or UGA

Stop codons are recognised by

release factors (proteins) - not tRNAs

Release factors (RF) bind to

stop codons, forcing the peptidyl transferase to catalyse the addition of water instead of an amino acid to the peptidyl-tRNA causing the complex to dissociate & the polypetide is released

RF1 recognises

UAA or UAG

RF2 recognises

UAA or UGA

Molecular mimicry

The structure of tRNA-EF complex is strikingly similar to the release factor

Ribosome recyling factor

Recycles ribosomes after completion of protein synthesis - Splits the ribosomes into subunits - Also requires EF-G

In eukaryotes termination is carried out by

a single release factor

Regulation of translation, cells can alter protein synethis due to

stress e.g. lack of growth factors, change in environment (temperature) or viral infection