midterm 4 bio99

proteins are

chains of amino acids joined together

how many different amino acids

20

amino group has

N

carboxyl group has

C

R group has

side chain, each AA has diferent R

each amino acid has ___ and ______ letter nickname

3, 1

bond that joins 2 AA togteher

peptide

prootein also known as

peptide

front of polypeptide, similar to 5’ on DNA/RNA

N terminus

back of polyoeptide, similar to 3’ on DNA/RNA

C terminus

adapter molecule

reads DNA or mRNA and translates to AA sequence

degenerate

multiple codopns encoden same AA

codon family

4 codons specify same AA in same box of chart

for wobble hypothesis

Anticodon Base (1st position, 5' end)	Can Pair With (Codon 3rd base)
G	U or C
C	G
A	U (standard only)
U	A or G
I (Inosine)	U, C, or A

inosine is

adenosine in wobble anticodon position of trna deaminated

genetic code read in

non overlapping triplets

if overlapping model was true

single base mutation would result in multiple amino acid changes in the polypeptide.

open reading frame steps

1. determned by stop codon

2. scnan ntil first AUG start codon

3. follow sequence 3 ases a a time until stop codon

each trna has this many “arms

4 to 5

anticodon arm

contains anticodon, interacts with mrna sequence

amino acid arm

attatches to amino acid

other arms

strucutral, interact with ribosome, trna synthetase

trna^tyr

trna that recognizes codon for tyrosie but doent necessarily have tyrosine aa attatched to it

tyr-trna^tyr

trna that recignuzes codon for tyrosuine and charged with tyrosine aa

aminoacyl-trna

trna with aa attatched to it

types of trna processing

1. base modification

2. cleavage

3. cca addition

4. introms removed

base modification

specific nucleotides modified

cleavage

ends of transcripts removed

cca addition

cca attatched to 3’ end of transcript, what aa attaches to

introns removed

only in eukaryotes

trna activation steps

1. adenylation

2. trna charging

trna activation steps catalyzed by

aminoacyl-tRNA synthetases (AATS),

trna activation step 1- adenylation

AA+ ATP to aminoacyl-AMP + PPi

AMP attatcheds to carboxyl group of AA

pyrophostape PPi generatedin rna, laterb hydrolyzed to 2 phosphates giving eenergy to make rxn irreversible

TRNA activation step 2- trna charging

aminoacyl amp+ trna= aminoacyl trna+amp

aminoacyl transferred off amp to trnas caa arm

2 classes of amino acyl trna synthesizes o trna charging different

class 1- attatch aa on 2’-oh of cca

class 2

attatch aa onto 3-oh of cca

what can aminoacyl trna synthetases do

aminoacyl trna synthetases distinguish between trnas

idenitty nucleotides

nucloetides of trna recongized by specific syntheases

scattered through trna

each synthase recongzes unique set of identity nucleotides

different trnas for same aa have same idenitty ucleotides and recog ized by same synthase

aa-trna proofreading cases

1. larger aa trying to attatch to smaller aa’s trna

2. small aa attahcing to larger aas trna

larger aa trying to attatch to smaller aa’s trna

amino acid binding site

• if aa big wont fit

• if aa small fits

small aa trying to attach to larger aas trna

Ile-tRNA synthetase has an acylation site,
as well as a separate proofreading site

Normal scenario: Isoleucine in Ile-tRNA synthetase

bacteria

initiator met trna

trna^fmet

bacteria

internal met trna

trna^met

transformylase converts ____ to __________

methionine to N formylmethionine (fmet)

fmet

formyl group attatches to nterminus of fmet, prevents fmet from attatching to aa in front of it, fmet can pnly be first aa

cant be added 9nternally

trna^fmet only one recongized by ribosome initiation complex

eukaryotes

initiator trna

trna_i ^met

eukaryotes internal trna

trna ^met

aminopeptidases

remove n terminal met in bacteria and eukaryotes

codon bias

some AA prefer specific codons and offer additional ways to regulate translation

DNA sequence can contain

• 5’UTR

• coding exons

• introns

• 3’UTR

types of mutations

1. single base substitutions

   1. silent

   2. missense

   3. nonsense

2. frameshift

3. deletion

silent mutation

change in codon doesn’t AA

missense mutation

change in codon results in aa

nonsens mutation

change in codon creates early spot codon

frameshift

insertion or deltion that alters reading frame, cant be in multiple of 3

deletion mutations

deletes 1+ nucleotides

transition mutation

1 purine subsituted for another purine (A to G or G to A)

most frameshift mutation result in

early stop codon

reversion mutation

acquiring second mutation to restore protein function

reversion mutation for missense

second mutation reverses back to original aa

reversion mutation for nonsense

changes stop back to aa

reversion mutation for frameshift

deletion of single nucleotide

translation process

1. processed mrna shuttled from nucleus

2. ribosome assembeld into mrna

3. trna with bound aa enters ribosome, base pairs with codon sequence on mrna

4. ribosome catlayzes formation of peptide bond between 2 aa bound to trna

5. ribosome moves along mrna 3 nucleotides at a time, new trnas enter and have aa attatched to growing polypeptide

are nucleotides or aa bigger

nucleotides

how many subunits do ribisomes have

2

small subunit and large subunit

how is rrna transcribed

as 1 long transceript

svedberg units (S)

roughly non linear relationship to size

based on sedimentation rates of ultracentrifugation

harry noller

looked at what is necessary for peptide bond formation

• rrna protein or both

puromycin

mimics trna, binds directly to large subunit, ribisomes attatche it to a growing polypepetide chain

protein denaturing compounds

unravels proteins structure

kethoxal

damages guanine in rna

ribozyme

rna with enzymatic activity (thomas steitZ)

how many trna binding sites does ribosome have

3

what are the trna binding sites of ribosome

• a site

• p site

• e site

a site

acceptor site, new trnas enter ribosome

p site

polypeptide sit, where growing polypeptide chain is held

e site

exit site, where trnas expelled after aa removal

which ribosomal subunit holds mrna

30s small subunit

which ribosomal subunit catalyzes peptide bond formation

50s large subunit

translation steps

1. aa-trna

2. initiation

3. elongation

4. termination

what makes puromycine a strong antibiotic

new aa can;t be added to puromycin so terminates translation

initiation basic steps

1. small subunit binds mrns

2. smal subunit binds initiator trna

3. large subunit binds to rest

initiation final product has

1. small subunit

2. large subunit

3. mrna (start codon at p site)

4. initiator trna (paired with start codon)

shine dalgarno sequence

consensus sequence in front of start codon

recruits small subunit to mrna

directs mrna start sire to correct positon on ribosom

kozak sequence

similar but slightly different function as shine dalgarno sequence in eukaryotes

if-1

fills a site to prevent trna from binding

if-2

escorts initiator trna

if-3

prevents large subunit from binding

translation initiation in bacteria

1a. blocking sites on small subunit

1b. leading mrna

2. loading initiator trna

3. loading large subunit

initiation in eukaryotes step 1

blocking sites on small subunit

similar to if1 bacteria, elf1a binds to and blocks a sire

similar to if3 in bacteria, elf3 and elf1 block large subunit from assembling

major difference from bacteria - mrna doesnt attatch at this step

initation eukaryotes

loading initiator trna

similar to if2 in bacteria, elf2 binds to initiator trna and escorts into p site of small subunit

elf2 bound to gtp

major differences from bacteria steo 2

elf5b- alos like if2 bound to gtp, joins initiator trna at p site

ststill no mrna

initiation eukaryotes step 3

loading mrna

elf4f- binds to 5’ cap of mrna and escorts to small subunit, no bacterial equicalent

3 factors

• elf4e- binds to 5’ cap

• elf4a-atpase and rna helicase

• elf4g- adapter, binds to elf3 and elf4e, links mrna to small subunit

requires hydrolysisof atp

mrna binds at 5’ cap not start codon

initiation eukaryotes step 4

scanning for start codon

scanning- complex travels along mrna until first start codon is found then stops

kozak sequence- helps identify start codon

initiation eukaryotes step 5

loading large subunit

both gtps hydrolyzed

drives release of all initiation factors

2 gtps requires

1 atp required to load mrna

large subunit can bind to pre initiation complex

elf4g

elf4g

part of elf4f complex, can bind to poly a binding (PABP)

most of eukaryotic translation called

cap dependent translation

internal ribosonak entry site (IRES)

sequence that can bind ELF4F and direct ribosome assembly and translation away from 5’cap

first identified in viruses that block cap dependent translation but allow their own genes to translate

some eukaryotic geens also use ires

makes eukaryotic polycistronic transcripts

bacterial polysomes

multiple ribosomes can translate same mrna simultaneously

translation elongation steps

1. aminoacyl trna enters ribosome

2. peptide bond forms

3. ribosome shifts to next codon

very similar between bacteria and eukaryotes