CH 11 The Central Dogma: DNA to RNA to Protein

What is the function of genes

To store information to make polypeptides

What is gene expression

A procedure to produce a functional protein using the information stored in the correlated gene

t or f - Genes are not able to perform any metabolic functions in the cell

true

What is the central dogma?

DNA-transcription-RNA-translation-protein

What is transcription

RNA is synthesized from DNA in nucleus

What is translation

Proteins are synthesized from mRNA in the cytosplasm

What is messenger RNA (mRNA)

an intermediate between a gene (DNA) and a polypeptide (Protein)

What does mRNA do

1. allows the cell to separate information storage from information utilization

2. allows a cell to greatly amplify its synthetic output

What is the role of RNA polymerase in transcription

1. responsible for transcription
2. incorporates nucleotides into a strand of RNA
3. binds to DNA at the promoter
4. requires the help of transcription factors to recognize the promoter

What is a promoter

The region of DNA where RNA polymerase binds and starts transcription

Functions of a promoter

1. determines which of the 2 DNA strands will be used a template
2. determines where transcription will begin
3. facilitates initiation of transcription

What are transcription factors

proteins that control the rate of transcription by binding to promoter region

What direction does RNA polymerase synthesize its RNA chain

5' to 3' direction (antiparallel)

How does RNA polymerase establish transcription bubble

1. DNA double helix reforms after RNA polymerase passes
2. Newly synthesized RNA dissociates with DNA template
3. DNA-RNA hybrid is about 9 base pair long

What are the 2 properties of RNA polymerase

1. is progressive - remains attached to DNA over long stretches

2. must be able to move from nucleotide to nucleotide

Describe transcription in bacteria

1. only 1 type of RNA polymerase in prokaryotes
2. composed of 5 subunits (core enzyme)
3. binds to random site on DNA
4. the sigma factor binds to the core enzyme to carry out interaction with DNA at specific promoter sites

Describe RNA polymerases in eukaryotes

1. requires transcription factors to recognize promoter
2. primary transcript aka pre-RNA
3. primary transcript will be processed to become mRNA
4. has a transcription unit which is the DNA segment to a primary transcript

What are the properties of mRNA

1. each mRNA codes for a specific polypeptide
2. found in cytoplasm
3. attached to ribosomes when translated
4. most have noncoding segment
5. eukaryotic mRNAs have specific changes at their 5' and 3' end

What specific modifications do the 5' and 3' ends have in eukaryotic mRNA

5': methylated guanosine cap
3': poly(A) tail composed of 50-250 adenosine residues

Functions of the 5' methylated guanosine cap

1. prevents 5'0end of mRNA from being digested by enzymes

2. aids in transport of mRNA out of nucleus

3. plays an important role in the initiation of translation

Function of 3' poly-A tail

protects the mRNA from premature degradation by enzymes

Where are all eukaryotic mRNA precursors synthesized?

produced in the nucleus by RNA polymerase II

How is the preinitiation complex (PIC) formed?

GTFs position RNA polymerases at transcription initiation sites

Describe PICs

1. assemble at the critical portion of promoter
2. lies 24-32 bases from initiation site
3. contains sequence of 5'-TATAAA-3' (named the TATA box)

What is TFIIH and what does it do?

It is an ATP-dependent helicase that possesses enzymatic activities

What does protein kinase do

phosphorylates RNA polymerase II

What does helicase do

unwinds double helix to generate transcription bubble

2 parts of primary transcript

exon and intron

What is an exon

a segment of a DNA or RNA molecule containing information coding for a protein or peptide sequence.

What is an intron?

a segment of a DNA or RNA molecule that does not code for proteins and interrupts the sequence of genes.

What is RNA splicing

removal of introns and joining of exons

What are splice sites

junctions of the introns and exons where mRNA precursor is cut and joined

Where is the splice site of 5' intron

GU

Where is the 3' splice site

AG

Describe the splicing machinery

1. pre-mRNA requires help from small nuclear RNA (snRNAs)
2. each intron becomes associated with spliceosomes
3. spliceosome has small nucleus ribonucleoproteins

What is alternative splicing

regulated process during gene expression that results in a single gene coding for multiple proteins

How are pre-mRNA transcripts processed?

1. cotranscriptionally
2. RNA transcripts associate w ribonucleoproteins
3. 5' cap and 3' tails are added
4. Introns are removed and exons are connected via RNA splicing

What are codons?

a genetic code that stores information in a gene

What are the 3 properties of a codon

1. triplets of nucleotides
2. non-overlapping
3. degenerate

Define "degenerate" in context of encoding genetic information

Some amino acids are specified by more than one codon

How do you identify codons?

1. first 2 codon bases for specific amino acid does not change
2. third codon base may vary
3. codons for specific amino acid tend to be clustered

What are the 3 types of single nucleotide mutation

1. synonymous
2. non-synonymous
3. nonsense

What is a synonymous mutation?

DNA mutation that does not change the amino acid sequence

What is a non-synonymous mutation

DNA mutation that causes an amino acid substitution

What is a nonsense mutation

DNA mutation prematurely destroys codon

What happens if one or more codon bases are inserted/deleted?

A frameshift DNA mutation alters the normal reading frame of the mRNA

What is the role of tRNAs in the context of decoding the codons

1. they translate mRNA codons into amino acid residues
2. they transfer amino acids to their specific codons

Describe the structure of tRNAs

1. has between 73-93 nucleotides
2. has significant percentage of unusual bases
3. all mature tRNAs end in CCA at 3' end
4. all tRNAs have complementary sequences

What are anticodons

A set of three nucleotides bases found on the tRNA. It is complementary to the codons on the mRNA.

Example of anticodon

The codon CGA on mRNA sequence codes for amino acid cysteine, tRNA corresponding to this codon is UCU anticodon.

What is the wobble hypothesis?

explains why multiple codons can code for a single amino acid

Wobble Hypothesis "Third Position"

U can be paired with A or G

G can be paired with U or C

I can be paired with U, C, or A

How are amino acids activated

1. specific aaRS link amino acids to their specific tRNAS

2. there are 20 different aaRS in an organism

Describe the structure of an eukaryotic ribosome

1. Has translation machinery
2. Has 3 tRNA binding sites

Describe the translation machinery of eukaryotic ribosome

1. Proteins - small subunit (40s) and large subunit (60s)
2. Ribosomal RNAs (rRNA) - structural support and catalyzes the linkage of amino acids to each other
3. Transfer RNAs (tRNAs) - translates info in mRNA nucleotide into amino acid

What are the 3 tRNA binding sites of eukaryotic ribosomes

APE
1. Aminoacyl site
2. Peptidyl site
3. Exit site

What are the required components for translation (7)

1. various tRNAs with attached amino acid
2. ribosomes
3. mRNA
4. proteins w different functions
5. GTO
6. Cations
7. rRNA

What are the 3 steps of translation

1. Initiation
2. Elongation
3. Termination

Explain step 1 initiation

1. initiation factors called elFs are required
2. the initiation codon is AUG (methionine)
3. Has 2 steps

2 steps of initiation

1. 43S complex binds to 5' of mRNA complex and searches for initiation codon (AUG)
2. Large subunit (60S) joins complex after 43S complex reaches initiation codon

What are the components of 43S complex

1. ribosomal 40S subunit
2. tRNA linked to methionine (AUG)
3. elF2-GTP

What are the components of mRNA complex

1. mRNA
2. elF4E that binds to 5'-cap
3. elF4A that removes double stranded structure
4. elF4G that links 5' to 3' end

t or f - The Kozak sequence defines the AUG codon: typically 5' CCACCAUGC 3'

true

What happens during step 2 of initiation

1. Large subunit (60S) joins the complex after 43S complex reaches the appropriate AUG codon
2. ALL eIFs are released
3. initiator tRNA binds to P site of ribosome

Describe step 2 (elongation)

1. the process of adding amino acid to growing polypeptide chain
2. specific proteins required (eEFs)
3. 2 GTPs required for each cycle
4. has 4 major steps

What are the 4 major steps of elongation

1. Aminoacyl-tRNA selection
2. Peptide bond formation
3. Translocation
4. Releasing the deacylated tRNA

Describe step 1 of elongation (aminoacyl-tRNA selection)

1. aa-tRNA binds to A site of ribosome


1. only the aa-tRNA with complementary anticodon can trigger conformational change of ribosome

Describe step 2 of elongation (peptide bond formation)

1. is catalyzed by peptidyl transferase
2. amino acids are brought to the ribosome by tRNAs and linked together to form a chain.
3. Once the matching tRNA has landed in the A site, the formation of the peptide bond that connects one amino acid to another begins.
4. This step transfers the methionine from the first tRNA onto the amino acid of the second tRNA in the A site.
5. results in a dipeptide bound to the tRNA at A site and tRNA in P site is deacylated

Describe step 3 of elongation (translocation)

1. ribosomes moves one codon in the 5' to 3 direction (moves to right) on the mRNA molecule
2. dipeptidyl-tRNA moves from A site to P site
3. Deacylated tRNA from P site to E site

Describe step 4 of elongation (releasing the deacylated tRNA)

The deacylated tRNA leaves the ribosome and empty the E site of the ribosome

Termination of protein synthesis in eukaryotes

1. Termination happens at 1 of 3 stop codons: UAA, UAG, UGA
2. Termination requires release factors


1. Eukaryotic cells have one class of release factor, eRF1, which recognize all the stop codons


3. Termination is accompanied by dissociation of the mRNA from the ribosome and the disassembly of the ribosome

What are polyribosomes/polysomes?

1. a complex of multiple ribosomes on mRNA that allows for simultaneous translation
2. polyribosomes/polysomes increases the rate of protein synthesis

Writing Question - What is the promoter? What is the function of the promoter?

The promoter is an area on DNA where RNA polymerase binds to before transcription begins. The promoter determines which DNA strand will be used as template and where transcription will begin. It also facilitates initiation of transcription.

Writing Question - Describe the five components of mRNA.

1. 5’ cap
2. 3’ polyA tail
3. Coding region
4. 5’ UTR
5. 3’ UTR

Writing Question - What is the function of the 5’ cap and 3’ polyA tail?

The 5’ cap prevents 5’ end of mRNA from being digested, aids in transport of mRNA exiting out of nucleus, and plays an important role in initiation of translation.

\
The 3’ polyA tail protects the mRNA from premature degradation.

Writing Questions - Explain the process of translation using the 3 main steps and what occurs in each step.

There are 3 steps: Initiation, Elongation, and Termination. In initiation, the 43S complex binds to 5’ end of mRNA complex and scans for the start codon (AUG). Then, 60S joins the complex after 43S reaches the AUG codon. In elongation, amino acids are added to a growing polypeptide chain. In termination, protein synthesis stops at either UAA, UAG, or UGA. Along with termination, mRNA disassociates from the ribosome and the ribosome disassembles.