DNA Replication, Transcription, and Translation Part II

genetic amplification

one DNA gene can produce many RNAs and each RNA can produce many proteins

RNA vs. DNA

linear polymer of four different nucleotides; made from sugar ribose; contains uracil (U) instead of thymine (T) that hydrogen bonds with adenine; single-stranded; can fold into a variety of shapes through internal base pairing; has enzymatic function as

What is true for both RNA and DNA?

They are both stabilized by hydrogen bonding.

transcription

RNA transcript becomes displaced from the DNA template; only a specific region is transcribed; carried out by RNA polymerase

RNA polymerase

catalyzes the formation of phosphodiester bonds in the 5' to 3' direction; moves down DNA helix unwinding the helix as it goes (
includes its own helicase activity
); uses ribnucleotide triphosphates (ATP, GTP, UTP, and CTP) as energy sources through hydr

RNA polymerase vs. DNA polymerase

links ribonucleotides not deoxyribnucleotides; can start transcription without a primer; RNA polymerase makes more mistakes

four types of RNA

tRNA (transfer RNA), mRNA (messenger RNA), siRNA/miRNA (small interfering RNA/small interfering RNA), rRNA (ribosomal RNA)

mRNA

RNA that is transcribed from genes in the DNA to be made into proteins; 1 eukaryote mRNA = 1 protein; in prokaryotes 1 mRNA can make multiple proteins

rRNA

a structural and catalytic RNA that forms the core of the ribosome; used during translation (protein synthesis); links amino acids in translation and catalyzes peptide bonds

tRNA

folded RNA that forms covalent bonds with amino acids to site of translation; carries amino acids to sites of translation

miRNA/siRNA

small hairpin RNAs that regulate eukaryotic gene expression; regulate the stability and degradation of mRNA to control protein expression

prokaryotic transcription

1. RNA polymerase binds to circular DNA and looks for promoter (specific sequence of DNA indicating the start site for transcription)
2. sigma factor (responsible for recognizing promoter) binds to RNA polymerase; RNA polymerase opens the DNA helix and st

similarities of DNA and RNA

same polarity (5' to 3'); hydrogen bonding between nucleotides; involved in central dogma; AGU coding, phosphodiester bonds, use polymerase for replication; negative charge because of backbone

differences between DNA and RNA

single vs. double stranded, T vs. U, deoxy vs. ribose, RNA enzymatic

prokaryotic promoters

conserved DNA sequences that direct RNA polymerase during transcription 10nt - 35nt long from start site upstream (start at 5'); governs direction of transcription (only 5' to 3') which can go in multiple directions depending on which template strand the

eukaryotic transcription

3 RNA polymerases and general transcription factors

RNA polymerase I

transcribes noncoding RNAs

RNA polymerase II

transcribes mRNA

RNA polymerase III

transcribes noncoding RNAs

general transcription factors

required for initiation and promoter recognition (TFIID and TBP)

eukaryotic transcription initiation

TATA box (DNA sequence rich in T and A) 25 nt from the start site upstream; requires general transcription factors
1. TFIID - transcription factors that contains the TATA box binding protein (TBP) which binds to promoter and serves as landmark/recognition

How do bacteria and eukaryotes transcribe and translate protein?

Bacteria transcribe and translate protein at the same time. Eukaryotes transcribe in the nucleus and translate in the cytoplasm.

pre-mRNA processing

addition of 5' cap
addition of 3' polyadenylation (polyA tail)

5' cap

occurs after 25 nt have been transcribed;
addition of 7-methyl guanosine triphosphate (modified nucleotide) to the 5' carbon of the first nucleotide of mRNA

3' polyA tail

addition of a series of repeated adenine nucleotides to the end of the mRNA; can be a few hundred nucleotides long

Purpose of 5' cap and 3' polyA tail

1. increase stability of the mRNA (doesn't get degraded as fast)
2. aid in the export from the nucleus
3. used during translation to make sure mRNA is fully mature
All of this takes place in the nucleus before mRNA is exported to the cytoplasm.

post-transcriptional RNA processing

in bacteria, most mRNA contain the uninterrupted coding region
in eukaryotes, genes contain both coding regions (exons) and noncoding regions (introns) called "pre-mRNA"
introns must be removed before mRNA can be translated; takes place by a process calle

spliceosome

how all noncoding introns are spliced out of a pre-mRNA; not all exons included in final mRNA

alternative splicing

selective inclusion or exclusion of exons; one pre-mRNA can make many different mRNA (thus different protein)

mature mRNA structure

5' cap
5' UTR (unstranslated region) i.e. noncoding sequence involved in regulating mRNA translation
exons (coding sequence)
3' UTR
3' polyA tail

three proteins important for regulating mRNA export and initiating translation

cap-binding protein (5' cap)
poly-A binding protein
EJC - exon junction complex (where empty introns are)

How do we code for each amino acid?

using exponents

ribosome (ribonucleoprotein)

a complex of ribosomal proteins and rRNA (ribosomal RNA) that contains a binding site for mRNA

composition of ribosome

large subunit which catalyzes the formation of the peptide bond and small subunit which matches the tRNA anticodon to the codons of mRNA

three tRNA binding sites

A site (addition) P site (peptide) E site (ejection)

A new tRNA binds to what site?

A site

ribozymes

RNAs that catalyze reactions

rRNAs in ribosome

make up core and do most of the catalytic function; contain a peptidyl transferase catalytic core

What is the start codon in translation?

AUG

tRNA in translation

80 ribonucleotides long that bind to amino acid and also bind to mRNA

two important regions to tRNA

anticodon: matching reverse compliment to codon; base pairs in the reverse complement direction with the codon of mRNA
3' CCA end

How many codons do cells have?

64

How many code for the M start translation?

1

How many code for the stop translation?

3

How many code for the 20 amino acids?

60

aminoacyl-tRNA synthetase

covalently couples each amino acid to its specific tRNA molecule; 20 unique ones for each amino acid;

codon

3 nucleotide sequence of mRNA that codes for an amino acid; there are 64 triplet codes that are available for more than 20 amino acids so some codes are redundant (more than one triplet can code for the same amino acid)

codons in translation

only one codon AUG (methione) codes for the start of translation; three codons signal terminaton of translation and do not code for any amino acid (UAA, UAG, UGA)

In what direction is mRNA translated?

5' to 3'

How many reading frames can mRNA have?

up to three

What are reading frames?

how the triplet code is read

translation initiation

1. small ribosomal subunit binds:
a. initiator tRNA in P site
b. carries methionine
c. additional proteins - translation initiation factors
2. loaded small subunit binds to 5' cap of mRNA
3. initiator tRNA and small ribosomal subunit scans the mRNA in the

What happens first during translation initiation?

Initiator tRNA, initiation factors, and small subunit binds together

translation

1. new tRNA is added to the A site; old tRNA is ejected from E site
2. peptide bond is formed between new amino acids in P and A sites (tRNA now holds polypeptide in A site)
3. large subunit moves forward on the mRNA (tRNA in P site is now in the E site a

release factors

proteins that bind to the A-site containing a stop codon; catalyzes the addition of a water molecule to the polypeptide causing release of it from the tRNA and the ribosomes dissociates from the mRNA
tRNA does not bind to stop codons!!!

How can we control gene expression?

mRNA production and degradation
protein production and degradation

proteases

enzymes that degrade proteins

proteosomes

a cylinder trash can complex of many proteases in eukaryotes; they act on proteins targets for degradation by the protein ubiquitin

What governs mRNA's gene expression?

the length of time it is allowed to exist

What happens when a mRNA is no longer needed?

it gets degraded by RNases

What are mRNA lifetimes like?

vary (minutes or hours); specified by specific 3' UTR of mRNA