Chapter 14 - From Gene to Protein

Transcription

process where genes are expressed as RNA

• info from DNA is directly used as a remplate to produce RNA

• uses complementary base pairing

  • Uracil (U) instead of Thymine (T)

translation

produces polypeptides from mRNA by translating codons

• turns information contained in a sequence of nucleic acids into amino acids

what is the link between genotype and phenotype

proteins

Archibald Garrod and what his hypothesis on genes and genotypes/phenotypes

• hypothesized that genes determine phenotype through enzyme production

• suggested that the symptoms of an inherited disease were due to the lack of the production of a specific enzyme

George Beadle and Tatum and metabolic defects

• broke genes of Neurospora by subjecting their cells under X-rays, and then added nutrients to the mold to find out which nutrients it now needed to survive

  • later identified that the specific enzyme that was lacking in each mutant functioned to produce arginine (an amino acid)

• created the one gene-one enzyme hypothesis

one gene-one enzyme hypothesis

• the function of a gene is dictated by the production of a specific enzyme

gene

region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule

• include noncoding portions (introns), promoters, and other regulator regions

introns

noncoding segments of DNA that are spliced off of mRNA before leaving the nucleus

exons

coding segments of DNA that code for protein production and stay on mRNA

• must be spliced together

The Central Dogma

• states that there is a directional flow to genetic information

• DNA → RNA → Protein

Ribosomes

site of translation of mRNA into a polypeptide

transcription and translation in bacteria

• transcription and translation in bacteria is mostly the same as in Eukaryotes

• main difference - lack of compartmentalization due to no nucleus

• transcription and translation happen concurrently (one after another, or at the same time)

transcription and translation in eukaryotes

• nucleus of eukaryotes separates transcription and translation

• pre-mRNA (primary transcript) is produced from DNA, which undergoes RNA processing to produce the final mRNA

• this provides extra control and variation in the final message

• mRNA exits the nucleus and is translated by ribosomes in the cytoplasm

how can 4 nucleic acids encode 20 amino acids

because translation requires using a triplet code

• 4 bases, so 4³ = 64 possible combinations

AUG

• start codon

• methionine

stop codons

• UAA, UAG, UGA

why does the mRNA code have redundancy?

each amino acid is encoded by multiple codons

• BUT no codon encodes for more than one amino acid

Transcription and DNA strands

• each gene uses only one strand of dna as its template strand for transcription

• different strands of the same molecule may be used as templates for different genes

• mRNA are complementary to their DNA due to base-pairing

• RNA is synthesized in an antiparallel, 5’ to 3’ direction similar to DNA replication

how is genetic engineering possible

because genes from one species are translated into the same proteins in another species

• genetic code is nearly universal in all species (we all use mostly the same amino acids)

small-scale mutations

mutations of one or a few nucleotides

• include substitutions or insertions/deletions

point mutations

changes in a single nucleotide pair

• can have drastic effect on protein structure and function

types of substitions

silent mutations, missense mutations, and nonsense mutations

silent mutations

type of substitution that does not change the coding for an amino acid

• no affect on phenotype

missense mutations

type of substitution which results in a different amino acid being coded for

• most common type of substitution

• may be beneficial, neutral, or detrimental

nonsense mutations

results in the insertion of a stop codon part way through a gene sequence

• almost always ends up producing a non-functional product

insertions and deletions

• 3 nucleotide-pair mutations may or may not lead to phenotypic alterations in structure and/or function of the gene product

  • reading frame is not altered in this type of mutation

frameshift mutation

alters the reading frame of mRNA

• insertion or deletion of a nucleotide (not in multiples of 3)

• entire codon sequence shifts causing large change in amino acids encoded for

• often leads to disastrous effects

• can be both missense and nonsense

transcription unit

• sequence of DNA which is transcribed into RNA

  • transcript works through copying specific units of DNA

upstream

• describes when a nucleotide sequence is located before another sequence

downstream

describes when a nucleotide sequence is located after another sequence

RNA polymerase (RNA pol)

synthesizes RNA from the DNA template during transcription

what does a DNA template require during transcription

promoter and terminator

promoter

region of attachment for RNA polynerase

ex. AUG start codon

terminator

signal which stops transcription

ex. UAA, UGA, and UAG stop codons

stages of transcription

1. Initiation - DNA unwinding and binding of RNA polymerase to the promoter

2. Elongation - RNA synthesized in 5’ to 3’ direction

3. Termination - transcript released and RNA polymerase dissociates

Initiation steps

1. small subunit binds to mRNA

• initiator tRNA always carries methionine

• initiator tRNA aligns with AUG start codon

2. Large ribosomal subunit joins to form the translation initiation complex

• assembly requires proteins called initiation factors and GTP hydrolysis

Elongation steps

1. Codon recognition

• amino acids are added to the growing polypeptide chain at the C-terminus

• aminoacyl tRNA anticodon pairs with the mRNA codon in the A site

2. peptide bond formation

• rRNA catalyzes peptide bond formation

• results in transfer of the polypeptide from the tRNA in the P site to the tRNA in the A site

3. Translocation

• mRNA translocates to align next codon in the A site

• empty tRNA moves to E site and is released

• tRNA with polypeptide moves into P site

Termination steps

1. ribosome reaches a stop codon on mRNA

• once reached, a release factor binds in the A site

• release factor is a protein that has a similar shape to tRNA

2. release factor promotes hydrolysis

• release factor causes hydrolysis of the bond between tRNA and the new polypeptide

• remaining components disassemble and are reused by the cell

what happens after translation

• folding of polypeptide occurs spontaneously

• may have addition of different molecules such as sugars, lipids, or phosphate groups to specific amino acids

• may have cleavage of amino acids from ends or cleaved into two or more smaller polypeptides

• may see multiple independently translated polypeptides coming together

• new polypeptides then move to specific structures/organelles

initiation in bacteria

• promoter contains the start point of RNA synthesis

• specific upstream sequences allow for RNA polymerase binding

  • also orient RNA polymerase in the proper direction

• multiple RNA polymerases may bind in the same direction

• RNA polymerase 1 directly recognizes these sequences and binds the template

initiation in eukaryotes

• TATA box - sequence of about 25 nucleotides upstream of the start site

  • contained in most eukaryotic promoters

• transcription factors mediate RNA polymerase 2 binding and the initiation of transcription

• transcription initiation

How is the bubble in elongation made during transcription

• 10-20 nucleotides are exposed as RNA polymerase moves along which allows pairing with RNA nucleotides

• as the RNA molecule elongates, it dissociates which allows the reformation of the DNA double helix

Termination in Bacteria

• termination sequence is transcribed, resulting in RNA polymerase detaching from the DNA and the new RNA transcript is released

• May be Rho dependent or independent

• the RNA transcript doesn’t need to be modified any further

termination in eukaryotes

• polyadenylation sequence (AAUAAA) is transcribed

• specific proteins bind to this sequence and then cut the pre-mRNA 10-35 nucleotides downstream

• pre-mRNA is further modified to produce the final mRNA

RNA modifications in Eukaryotes

• pre-mRNAs need to go through RNA processing before leaving the nucleus for translation

• both ends of the primary transcript are altered, certain interior sequences are removed, and the remaining sequences are then spliced together

End modifications (2 types)

• 5’-cap - composed of a modified guanine nucleotide, is added after the first 20-40 nucleotides are synthesized

• poly-A tail - added after the polyadenylation signal at the 3’ end

roles of end modifications

• facilitate export from the nucleus

• Protect mRNA from enzymatic degradation in the cytoplasm

• Aid in ribosome attachment

RNA splicing

• introns are removed

• Exons are spliced together

• Untranslated regions (UTRs) at 5’ and 3’ ends are not removed as they contain important binding and regulatory info

• RNA splicing results in a continuous mRNA molecule containing the coding sequence

Alternative RNA splicing

• Uses different segments as exons during splicing

• Produces different polypeptides from a single transcript

• Results in more products than number of genes in the genome

spliceosome

• Removes introns and joins the ends of exons together

• binds to specific sequences at the ends and within introns

• Consists of multiple proteins and small RNAs (ribozymes)

Ribozymes

RNA molecules which function as enzymes

Its characteristics allow for the catalytic activity of RNA:

• base pairing within an RNA molecules results in a 3D shape

• RNA contains functional groups that can function in catalysis

• Hydrogen bonding adds specificity of interactions

tRNA structure

• contains an amino acid attachment site and an RNA binding recognition site (anticodon)

• Intramolecular base pairing (which creates the loops in its shape)