MMBIO 240 - Final Exam

ribosomal RNAs (rRNA)

structure and function of ribosomes; ribosomes perform translation of proteins from mRNAs; enzymic activity to help chemical reaction of peptide bond formation in growing chain of amino acids

eukaryotic 80S ribosome

large 60S and small 40S subunit

sedimentation rate

S, a measure of shape and size of molecules

peptidyl transferase activity

links new amino acid to peptide chain; ribozyme

transfer RNAs (tRNA)

bring amino acids to ribosome for protein production; decipher genetic code by matching anticodons in tRNA to codons in mRNA

initiator tRNA

recognizes start codonsc

charged tRNA

carries amino acid to ribosome to contribute to protein synthesis

steps for mature tRNA

cleave 3‘ end and end with CCA and 3‘ OH, trim 5‘ end and end with GGG and 5‘ monophosphate, remove introns, modify to make unusual bases, add CCA sequence to 3‘ end in eukaryotes, charge by adding amino acid via aminoacyl tRNA synthetase to hydroxyl on ribose in A of CCA at 3‘ end

unusual bases

NOT incorporated by RNA polymerase, AFTER transcription, present in loops and protect from degradation

EPA sites

empty tRNA site, peptide chain mostly near peptidyl tRNA, A site holds Aminoacyl tRNA aka acceptor site if codon matches

human rRNA genes

active translation in nucleus, 200 rRNA transcription genes total across five chromosomes; not all actively used and active genes appear to be naked DNA and have no nucleosomes

10S, 5.8S, and 28 S rRNAs

transcribed as pre rRNA by RNA polymerase I; ETS and ITS excised to make mature rRNAs, like tRNA maturation not like intron splicing

intergenic spacers of rRNA

many promotors and enhancers

5S

encoded separately in genome, transcribed by RNA pol III

pre rRNA to mature rRNA

trimming of ETS and ITS; add methyl groups to ‘ OH; conversion of some bases to pseudouridine; remove introns by internal ribozyme

small nucleolar RNA (snoRNA)

localized to nucleolus; modify rRNAs through methylation and pseudouridine formation

snoRNP

snoRNA bound to protein

direct recognition of which bases to add methyl groups to and which bases to convert to pseudouridine

prokaryotic ribosomal assembly

add purified rRNA and protein to test tube and ribosomes self assemble

eukaryotic ribosomal assembly

“chaperone” proteins required for assembly, not part of final product; ribosomal structure conserved through all domains of life

nucleoulus

transcription of pre rRNAs by RNAPI and RNAPIII, maturation to make rRNAs, formation of pre 90S immature particle with ribosomal and non ribosomal proteins

nucleoplasm

formation of pre 40S and pre 60S subunits; most non ribosomal proteins shed and rRNA is trimmed

cytoplasm

exported; final maturation, including rRNA trimming; shedding of final non ribosomal proteins

rna polymerase III

short RNAs

55 rNA, tRNA, U6 snRNA, HI RNA

removes 5’ leader region from tRNA

upstream of transcription start, type one and type two RNAPoI III promotors are internal to the gene; initiation complex is three to four subunits

RNA polymerase III transcription

Multiple types of promotors, small untranslated transcripts

transcription location

free ribosomes in cytoplasm and ribosomes on rough ER

microsomal fraction

protein synthesis; can purify organelles based on density; isolate ribosomal components; includes rough and smooth ER

types of proteins

destined to stay in cytoplasm or nucleus; destined to stay in organelles; intended to exit to cell; inserted into various cell membranes

proteins destined for cytoplasm or nucleus translated on free ribosomes

proteins destined for other locations translated on ribosomes on rough ER so proteins can be sent to secretory pathway after translation

crick’s adaptor hypothesis

at least twenty different adaptor RNA, one for each amino acid; amino acid attaches to cognate adaptor before protein incorporation

CCA

all charged tRNA at three prime end; conserved in tRNA for prokaryotes; not conserved in tRNA in eukaryotes and must be added later by tRNA nucleotidyl transferase; critical for activity

acceptor stem

site of amino acid addition, at 3’ end

anticodon

site that base pairs with mRNA to confer specificity to matching genetic code

length conservation

length of acceptor, anticodon and T C arms conserved; length of D arm mostly conserved, variable arm differs the most

animoacyl tRNA synthesis

add amino acid to ATP and drop pyrophosphate; transfer amino acid to tRNA molecule; twenty, one for each amino acid, each can charge multiple types of tRNA molecules

genetic code

prediction of what amino acids produced on translation of particle rmRNA sequence; organized into codons; mostly universal

all acidic amino acid codons start with GA; all charged amino acids have A or G in second position; all amino acids with U in second position are non polar

each base is one codon and one codon consists of three bases, single amino acid

no intervening bases between codons; all bases used between start and stop codons

code highly degenerate, same amino acid encoded by multiple different codons

insertion and deletion mutants

reveal genetic code is based on triplet codons

poly U

UUUUUUU encodes polyphenylalanine

poly A

AAAAAA encodes polylysine

poly C

CCCCCCC encodes polyproline

poly G

did not work, forms a triple stranded helix

polyribonucleotides

ribosomes in presence of charged tRNA to see what proteins are produced

sequences direct synthesis of polypeptides of polypeptides

trinucleotide binding assay

trinucleotides and charged tRNAs pass through filter, ribosomes, UUU, and Phe tRNAs form complex and get stuck

reading of genetic code

mRNA read five prime to three prime, protein produced in N to C terminal direction

start codon

AUG, encodes sulfur containing methionine

stop codon

UAA, UAG, UGA; only stop translation if in right reading frame; DNA mutations resulting in insertion or deletion cause stop codons earlier

open reading frame

sequence from start codon to stop codon

reading frame

every mRNA sequence has three frames due to triplet codons

wobble hypothesis

codon and anticodon form antiparallel base pairs; first two bases in codon form standard Watson Crick base pairs with last two bases in anticodon; third base of codon and first base of anticodon form nonstandard base pairs; allows some aminoacyl tRNAs to bind to more than one codon

bacterial initiation

utilizes fMet and initiator tRNA; formal group on methionine

modified tRNAmet: initiation; used at N terminus

regular tRNAmet: elongation

IF1

bind to A site and prevent tRNAs coming in until ready

IF3

keep 50S and 30S subunits from prematurely reforming 70S and only allow initiator tRNA in

IF2

bind to initiator tRNA and hydrolyzes GTP and GDP

steps in translation

30S subunit binds fMet tRNAfMet and mRNA, and then 50S subunit joins

IFs keep ribosomal subunits separate until start codon and initiator tRNA are recruited; ribosome splits at termination

translation in prokaryotes

16S rRNA base pairs with Shine-Dalgarno sequence to help ribosome find right site for translation initiation

eukaryote scanning mechanism

initiator tRNA base pairs with a start codon that is surrounded by a Kozak sequence to help ribosome find right site for initiation

kozak sequence

conserved and helps ribosomes find the right AUG to start translation, found by initiator tRNAs

alternative start codons

some genes encode multiple start codons which increases diversity of proteins produced from a single gene

elongation factors

bring charged tRNA molecules to ribosomes and ensure correct ones are matched to mRNA codon; help translocate mRNA through ribosome

prokaryotes

EF Tu, EFIB, EF G

EF Tu: binds aminoacyl tRNAs and GTP and test for match in A site

EF G: translocation; moves A site tRNA to P site, then P site tRNA to E site

eukaryotes

eEFIA, eEFIB, eEF

release factors

proteins recognize stop codons through protein:RNA interactions, no internal RNA, release peptide chains from ribosome; composed of amino acids not RNAs and “tripeptide codons” try to bind in A site, and bind to anticodons

RF1

recognizes UAG and UAA stop codon

RF2

recognizes UGA and UAA stop codon

RF3

enhances activity of other RF factors to release peptide chain from ribosome and helps exit A site

ribosomal recycling factors

break apart small and large ribosomal subunits