Insertion/Deletion of U residues 

Enzymatic deamination of A to I or C to U

Forms a capped RNA

2 exons from different primary transcripts joined

Step 1) OH on pre-mRNA attacks 5' splice site of slRNA

step 2) free OH at 5' splice site attacks 3' splice site, forming capped RNA

U residues inserted or deleted

Happens in mitochondria

Uses editosome to catalyze and guide RNA (complementary to target RNA) 

Editosome cuts near mismatch and U residues are added

Editosome

Catalyzes RNA editing

has TUTase which ensures only U residues are added

Has small guide RNAs which are partially complementary to the target RNA

Guide RNA

Partially complementary to target RNA in editing

3 regions:

Anchor-complementary to target RNA

Middle-contains editing info

3' U extension

Substitution RNA editing 

Uses deamination of As or Cs

ADAR edits the RNA, catalyzes A to I conversion

Cytosine deaminase edits RNA, converts C to U

Adenosine deaminase 

Catalyzes A to I

ADAR likes double stranded RNA

Edits BEFORE splicing

editing enzyme (like ADAR)

Do C to U conversion

critical for gene expression

Catalyzed by ribonucleases

Complete hydrolysis of RNA into component nucleotides

Rates very

Once RNAs are processed, they are exported to cytoplasm

Splicing puts proteins (Exon junction complex) on mRNAs that go with them to cytoplasm

These proteins should be removed in 1st rd of translation

Triggered by exon junction (protens that attached during splicing)

If exon junction complex proteins arent removed during translation, decay is triggered on that RNA

Targets mRNA without a stop codon

Ribosome translates to poly A tail, stalls at end of tail, protein and mRNA degraded

RNase P processes 5' end of tRNA

RNase D processes 3' end of tRNA

Introns may need to be removed too

CCA added by enzyme nucleotidyltransferase 

tRNA bases are then methylated

RNase P

Used in tRNA processing 

Processes 5' end of tRNA

Need RNA component, not really protein part

RNase D

Needed for tRNA processing

Processed 3' end of tRNA

Added CAA to tRNA

Boinds 3 nucleotides to catalyze formation of CCA without template

rRNAs are cut out of larger transcipts

For bacteria, transcipts have the same rRNAs, but different tRNAs

For eukaryotes, POL I makes the transipt that 3 rRNAs come from

snoRNPs process rRNAs

Protein coding region of mRNA

Combination of 3 nucleotides

Codes for move from RNA to protein

Hypothesized on Adaptor Molecule, which he thought would recognize certain codons from mRNA, and each adaptor would carry a certain amino acid

This Adaptor Molecule turned out to be tRNA

small RNA molecule that covalently links to amino acids

Adaptor Molecule

Codon of mRNA and anticodon of tRNA base pair antiparallel

Change the codon to protein sequence

75-95 bases long

Ends with CCA at 3' terminus, which is where the amino acid attaches

tRNA and attached amino acid

Produced by aminoacyl-tRNA synthetase enzyme

Colverleaf structure 

4 Arms

Acceptor Arm where amino acid links

D Arm which contains dihydrouridine

Anticodon Arm which base pairs with complementary codon in mRNA

Pseudouridine Arm, which has weird Carbon-Carbon bond between base and ribose

Several code words have same meaning (multiple codons make same amino acids)

61 codons for coding amino acids

3 for termination of translation (stop codons)

1st 2 nucleotides are the same, the last is different 

Codon family-4 codons for same nucleotide

Wobble Bases-the base that pairs to many nucleotides

Wobble

As long as 2 nucleotides are right, the 3rd can vary so that 1 tRNA can pair to more than 1 codon

Its the 3' nucleotide on the codon that can vary

Wobble bases-The base that can pair to various nucleotides

is the 5' nucleotide on the anticodon

Very flexible Wobble base

Can pair with A, U, or C

On the 5' anticodon position

Each triplet in mRNA

Depends on where translation appartatus starts

Amino acid sequence  of protein depends on which reading frame is used 

Start Codon-At AUG, sets reading frame, starts translation

Stop Codon-Stops translation, UAA, UAG, UGA

Long sequence without a stop codon

Encodes a protein 

Nonsense Mutations

Aborts protein synthesis

Causes incomplete protein

Mutation resolved with Suppresor tRNAs

Mutant tRNAs at stop codon that allow translation to continue

Correct nonsense mutations

Suppression shouldnt be too efficient, or it would unstop too many stop codons

Multiple tRNA genes resolves this

Crick

In experiments, if genetic code overlapped, a single-nucleotide change would alter 3 codons, so 3 amino acids would change

If no commas, frameshift mutations would throw off whole reading frame, but if there were commas, frameshift mutations would only change 1 amino acid

If codons read in sets of 3 nucleotides, crossing 3 B-gene mutants with same sign would re-establish reading frame 

Synthesized RNA templates and cell free extracts used to crack code

Poly U directed synthesis of Poly Phe (1st discovery)

We use codons our tRNA works best with

Ribosomes

Translation happens on ribosomes

Ribosomes responsible for protein synthesis

Made up of 50s and 30s subunits

rRNAs and rProteins spontaneously form ribosomes 

Association of ribosomes at start of protein synthesis and dissociation on release of completed polypeptide is fundamental to translation

Each ribosome makes 1 polypeptide, but multiple ribosomes can occupy each mRNA during translation 

rRNA catalyzes protein synthesis and peptide bond formation

rRNAs are driving force behind most ribosome interactions

Ribosomes have binding sites for 3 tRNAs

APE

A-for aminocyl-tRNA binding

P-Peptidyl-tRNA binding

E-Exit site

All spots span the 2 ribosomal subunits and link them. They also position the tRNAs for bond formation

Requires formation of acyl linkage between amino acid and tRNA

Amino acid-tRNA synthetases activate amino acid before it attaches to its tRNA 

2 Steps: Adenylylation and tRNA Charging 

Step 1 of Charging:Adenylylation

Aminoacyl transferred to tRNA

THe amino acid can be transferred to either 2' OH or 3' OH of the tRNA A site depending of the type of synthetase

A certain tRNA synthetase is responsible for attaching each of the 20 amino acids to its tRNA, but each can recognize multiple tRNAs 

Each synthetase specific for certain amino acid and certain tRNAs, 2nd genetic code

the Synthetase looks at nucleotides in amino acid acceptor arm and anticodon arm

10 if more specific nucleotides involved in recognition of tRNA by the synthetase

Attachment of correct amino acid to the tRNA is essential bc ribosome doesnt check this

Initiation

Translation

ELongation

Protein synthesis always proceeds from amino to carboxy terminus

Most regulated step
3 steps

1) Recruitment of small ribosomal subunit to mRNA

2) Indentification of start codon

3) Recruitment of large ribosomal subunit

Start codon codes for Met

All organisms have 2 tRNAs for AUG to Met

1 for when AUG is initiation codon

1 for when its in the middle of reading frame

3 initiation factors needed: IF1, IF2, IF3

Translation begins with binding of ribosomal small subunit to an mRNA

Initiation guided by base pairing between 16srRNA-mRNA

Initiator tRNA carries N-formylmethionine

mRNA-16snRNA

Shine-Dalgarmo site

Initiation signal thats 4-9 bp long, located 8-13 bp uptream of start codon

Attracts mRNA

Positions the ribosome

Cap recuits the small ribosomal subiunit to mRNA. The small subunit scans for AUG start codon.

Kozak sequence enhances translation

 Process:

The ribosomal subunits kept seperate by initiation factors eIF3 and eIF1A, the functional homologs of IF3 and IF1.

eIF3 prevents early subunit joining

eIF1A blocks tRNA at A site

eIF1 binds to E site

eIF2, GTP, and charged initiator tRNA form a 43S initiation complex, along with eIF5 and eIF5B

Binding of this 43S initiation complex is mediated by eIF4 complex which contains:

eIF4E binds 5' cap on mRNA

eIF4A is an ATPase and RNA helicase

eIF4G which binds eIF4E and eIF3 to maje bridge betwn 43S complex and mRNA

This large 43S,eIF4F, mRNA complex scans, looking for start codon.

Once start codon found, eIF1, 1A, 2, and 3 are kicked off so 60S subunit can join

eIF5 and eIF3 help kick it off

A site is empty, P site is assembled, synthesis can begin

Step 1

:30S subunit binds IF1 to IF3. 

IF 1 binds to A site to bloack tRNA binding.

mRNA binds to 30S subunit at ribosome binding site, which puts start codon at P site (the only spot initiator tRNA can bind.)

During elongtion, all new tRNA must go to A site 1st

Step 2

30S complex joined by GTP bound IF2 and initiating tRNA.

Anticodon of this tRNA pairs with the mRNAs initiation codon at P site.

Step 3

Change in30S subunit triggers release of IF3, so now 50S subunit can join.

The GTP cound to IF2 is hydrolyzed, then IF2 and IF1 released.

All these form a functional 70S ribosome initiator complex with initiator tRNA at P site

EF-Tu

EF-Ts

EF-G 

Need GTP

Incoming aminoacyl-tRNA binds first to a complex of GTP-bound EF-Tu, then to the A site of the 70 initiation complex. GTP hydrolyzed and EF-Tu-GTP complex releases the tRNA. 

To ensure accuracy, 2 adjacent As in the rRNA provide extra bonding with correctly paired bases, GTPase only works when tRNA is placed correctly, and tRNAs must be rotated for peptide bonds (incorrectly paired tRNAs cant do this) 

Peptide bond formed between 2 amino acids bound by thier tRNAs to the A and P sites on ribosome. This is catalyzed by the 23S rRNA of large subunit. 

Ribosome acts as ribozyme

2'OH group on 3' S in the P site of tRNA also important

Elongation Process (Step 3) Translocation

After peptide transferase, the tRNA at the P site is empty

The growing chain is linked to the tRNA at the A site

The P site tRNA moves to the E site and the A site tRNA moves to the P site. A site empty again

mRNA moves 3 bases

The ribosome translocated separately. The large subunit goes 1st. The tRNAs 3' ends have moved but not the anticodons

Movement of the ribosome requires energy from hydrolysis by EF-G. With a GTP, the EF-G binds the ribosome. A peptide transferase and shift give binding site for EF-G (like a charged tRNA). EF-G translocateds A site tRNA. Now empty site, ready for next tRNA. 

Class 1 release factors

Recognize termination codons and trigger hydrolysis of the terminal peptidyl-tRNA bond from the tRNA P site

RF-1 UAG and UAA

RF-2 UGA and UAA

eRF1 in eukaryotes, does all 3

Release class 1 factors from the ribosome after release of chain accomplished

RF3 and eRF3

Regulated by GTP binding and hydrolysis

After Termination, ribosomes are removed from the mRNA by ribosome recycling factor, which recruits EF-G to stimulate release of the uncharged tRNAs in the P and E sites

Eukaryotes have no RRF, must dissociate differently

Inhibit protein synthesis by targeting differences between eukaryotic and bacterial systems

Puromycin

Cycloheximide

Occur when transciption ends early

Ribosome reaches end of mRNA and stalls

tmRNA recognizes this problem

Looks like a tRNA charged with Alanine and an mRNA

Binds to a site of stalled ribosome

Acts in place of mRNA

Codes for 9 amino acid and stop codon, which signal for termination

Eukaryotes

No stop codon on mRNA

Mediated by protein Ski7, relative of eRF3

Needs exon junction complex formed during splicing

First round of translocation displaces them

If not, it triggers decay

Expression of genes must be regulated so their products are present in thr right amt and when needed

Gene expression can be regulated at many different pts in synthesis of RNA and proteins

Regulation at transcription is most common

Changes in how RNA polymerase interacts w/promoter sequences

Difference in promoter strength correlates with how efficient the genes are transcribed or how well RNA poly "sees" the promoter

Constituitive genes

Housekeeping genes

When level of a gene product rises and falls w/cells needs

Regulatory proteins-Alter affinity of RNA poly for promoter

-Also called transcription factors

-Recognize specific DNA binding proteins at genes they control

-Some work from distance due to loops (esp in humans)

Regulatory proteins that enhances gene expression by helping recruit RNA polymerase to promoter

Helps with open/closed promoter complexes

Distant regulatory sites

Can be enhancers or repressors

Control of a gene by multiple regulators

1 specific/1 general

Has promoter that controls transcription for all genes

Bacteria only

1 poly RNA has several ORF for initiation

Group of operons w/ common regulator

Results in global regulation

Recognition of DNA by regulatory protein through amino acids of a alpha helix

Reads along major grove

Short inverted repeats

Usually dimmers

Bind for regulatory proteins

Bacterial regulators

2 short alphas connected by beta turn

part of DNA binding domain

Homeodomain proteins

60 amino acid seq

Regulate development in fruit flies and humans

Has helix turn helix

Dimerization domain

Positions 2 recognition helices

Has hydrophoebic amino acids as surface for dimerization

 Basic leucine zippers

30 amino acids forming loop held together by zinc ions

Coordinated by 4 Cys or 2 Cys and 2 His

Presents recognition helix to DNA

Showed DNA binding and activation domains funcitonally seperate

Activation domain fused w/DNA binding domain from bacterial repressor

If repressor DNA binding site present, activator worked

Best model of gene regulaiton in bacteria

Jacob and Monod did it

1 enzyme involved in lactose metabolism was inducible

The enzyme was B galactosidase, which was easy to assay

Mutants in the B gala gene werent able to grow on lactose as carbon source

Other mutants that were lac were found, there couldnt transport lactose into the cell

The mutations defined a new gene lacY which codes for protein that brings lactose into the cell

The operon is transcribed as a single unit 

The RNA has 3 ribosome binding sites

Mutants produced all 3 gene products

Jacob/Monod isolated these a performed merodiploid analysis, which showed there were 2 types of constituitive mutants for the lac operon: LacI and Oc

Complemented or rescued by merodiploid

When a normal gene masks the prescence of a defective it is complementaiton

LacI mutations are recessive

They can be complemented int rans

A gene that was coding for a protein that regulated the rest of the genes of the lactose operon

This is a regulatory gene-its gene product repressed expression 

Defined Cis acting sites

Could not be rescued/complemented in merodiploid

Sequences that fuctioned only as seqs, never transcribed or translated, LacO defined a region where the LacI gene product would bind to the DNA and repress 

Control involves 2 elements

1) protein repressor (LacI)

2) DNA seq (operator site)

Responds to presence of lactose

Gene is not part of operon

Binds to the opeator site in absense of lactose and blocks transctiption

In presence of lactose, sm amt added

Acts as effector molecule

Inducer

Binds to repressor and causes loss of affinity for DNA 

Operon then becomes active

GLucose

E COlis preferred energy source

Cell isnt interested in metabolizing other Carbon source is glucose is there

mediates effect of glucose

cAMP-CRP binds to a site near promoters, causes transcrition w/glucose involved

CRP-positive regulator 

Lactose promoter doesnt work unless CRP present

Codes for 5 genes that encode to make amino acid tryptophan

Expressed only when trytophan is limiting

E coli can make its own amino acids

TRP repressor controls transcription of operon

When Try. present, repressor active, so Try. is a co-repressor

Regulates gene exp. over 700 fold range, 10x more than repressor

Key is leader sequence of trp RNA

2 weird features: Leader peptide, and Rho ind terminator

Regions 3 and 4 base pair to form Rho ind terminator (GC rich, run of Us) 

Regions 3 and 3 can pair, which doesnt form terminator

Translation of leader peptide determins hairpin choice

If translated right, terminator forms

has 2 back to back trp codons

if no trp-tRNAs, no terminator forms

Regulation by RNAs

Riboswitches

All regulators are proteins

Riboswitches are examples of CIS acting RNA regulatory elements

RNAs assume different shapes which affects expression

Respong to small molecules

Located within 5' untralated region of the genes they control 

Made of:Aptamer and Expression platform

Aptamer-Binds small molecules, undergoes change, causes change in expression platform