MCAT Biochemistry - RNA and the Genetic Code

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central dogma of molecular biology

major steps involved in the transfer of genetic information

gene

a unit of DNA that encodes a specific protein or RNA molecule; can be expressed through transcription and translation

direction of transcription

5’ to 3’

direction of translation

5’ to 3’

Messenger RNA (mRNA)

carries the information specifying the amino acid sequence of the protein to the ribosome; transcribed from template DNA strands by RNA polymerase enzymes in the nucleus; may undergo a host of posttranscriptional modifications

codons

three-nucleotide segments that designate a specific amino acid

monocistronic

each eukaryotic mRNA molecule translates into only one protein product

polycistronic

prokaryotic mRNA may code multiple proteins; starting the process of translation at different locations in the mRNA can result in different proteins

Transfer RNA (tRNA)

responsible for converting the language of nucleic acids to the language of amino acids and peptides; contains a folded strand of RNA that includes a three-nucleotide anticodon that recognizes and pairs with the appropriate codon on an mRNA molecule; found in the cytoplasm

charged/activated tRNA

connected to an amino acid at the 3’ end by aminoacyl-tRNA synthetase

aminoacyl-tRNA synthetase

each amino acid is attached to tRNA at the 3’ end (CCA sequence) with this enzyme; requires two ATP; bond supplies energy for peptide bond

Ribosomal RNA (rRNA)

synthesized in the nucleolus; functions as an integral part of the ribosomal machinery used during protein assembly in the cytoplasm; helps catalyze the formation of peptide bonds; splices out its own introns within the nucleus

ribozymes

enzymes made of RNA molecules instead of peptides

Genetic code tables

easy way to determine the amino acid that is translated from each mRNA codon (4³ or 64 codons); unambiguous - each codon is specific for one and only one amino acid; degenerate - multiple codons code for each of 20 amino acids; universal - same across all species

anticodon

on tRNA; recognises codon on mRNA; antiparallel. orientation

start codon

signals the beginning of translation

methionine (AUG)

stop codons

encode for termination of protein translation; no amino acids on the associated tRNA

UGA, UAA, UAG

degeneracy

more than one codon can specify a single amino acid (except methionine and typtophan)

wobble position

for most amino acids, the first two bases are usually the same, and the third base in the codon is variable; protect against mutation

silent/degenerate mutation

no effect on the expression of the amino acid and therefore no adverse effects on the polypetide sequence

point mutation

affects one nucleotide in a codon

expressed mutations

can affect the primary amino acid sequence of the protein

Missense mutation

one amino acid substitutes for another

Nonsense (truncation) mutation

the codon now encodes for a premature stop codon

reading frame

three nucleotides of a codon

frameshift mutation

some number of nucleotides are added to or deleted from the mRNA sequence, shifting the reading frame, usually resulting in changes in the amino acid sequence or premature truncation of the protein; typically more serious than point mutations

Cystic fibrosis

most commonly caused by a frameshift mutation; results in a defective chloride ion channel that can’t reach cell membrane, leading to blocked passage of salt and water into and out of cells; cells that line the passageways of the lungs, pancreas, and other organs produce an abnormally thick, sticky mucus that traps bacteria, increasing the likelihood of infection in patients

transcription

creation of mRNA from a DNA template

helicase

unzips/opens DNA during transcription

topoisomerase

releives tension in DNA strands during transcription

template (antisense) strand

antiparallel and complementary mRNA synthesized from this strand

promoter regions

RNA polymerase locates genes by searching for specialized DNA regions

RNA polymerase II

transcription enzyme in eukaryotes; produces hnRNA and small nuclear RNA (snRNA); doesn’t need primer; read 3’ to 5’, buikds 5’ to 3’; promoter region: TATA box

TATA box

binding site for RNA polymerase II in transcription; named for its high concentration of thymine and adenine bases

Transcription factors

help the RNA polymerase locate and bind to this promoter region of the DNA

RNA polymerase I

located in the nucleolus and synthesizes rRNA

RNA polymerase III

located in the nucleus and synthesizes tRNA and some rRNA

coding (sense) strand

not used as a template during transcription; identical to the mRNA transcript except that all the thymine nucleotides in DNA have been replaced with uracil in the mRNA molecule

base numbering system

identify the location of important bases in the DNA strand relative to a gene

first base transcribed = +1

upstream = negative

downstream = positive

no zero

TATA box ~ -25

heterogeneous nuclear RNA (hnRNA)

unprocessed mRNA

Posttranscriptional Processing

Intron/exon splicing

5′ cap

3′ poly-A tail

introns

noncoding sequences

exons

coding sequences

spliceosome

large snRNP/snRNA complex found primarily within the nucleus of eukaryotic cells that removes introns; recognizes both the 5′ and 3′ splice sites of the i; excised via lariat and degradedntrons

small nuclear RNA (snRNA)

a class of small RNA molecules that are found within certain complexes of the cell nucleus in eukaryotic cells

small nuclear ribonucleoproteins (snRNPs)

forms a complex with snRNA to make spliceosomes

lariat

lasso-shaped structure

7-methylguanylate triphosphate cap

5’ end of hnRNA; added during the process of transcription; recognized by the small eukaryotic ribosomal subnunit as the binding site; protects the mRNA from degradation in the cytoplasm

polyadenosyl (poly-A) tail

3’ end of hnRNA; protects the message against rapid degradation; assists with export of the mature mRNA from the nucleus

Untranslated regions (UTRs)

still exist at the 5′ and 3′ edges of the transcript because the ribosome initiates translation at the start codon (AUG) and will end at a stop codon (UAA, UGA, UAG)

alternative splicing

the primary transcript of hnRNA may be spliced together in different ways to produce multiple variants of proteins encoded by the same original gene

nuclear pores

hole sin the nuclear membrane mRNA leaves through

translation

converting the mRNA transcript into a functional protein; requires mRNA, tRNA, ribosomes, amino acids, and energy in the form of GTP

ribosome

composed of proteins and rRNA; composed of large and small subunits that only bind together during protein synthesis; bring the mRNA message together with the charged aminoacyl-tRNA complex to generate the protein; three binding sites in the ribosome for tRNA: APE

eukaryotic ribosomal subunits

28S, 18S, and 5.8S rRNAs gens in nucleolus → 28S, 18S, and 5.8S rRNAs transcribed by RNA polymerase I in one unit = 45S → processed into 18s rRNA of 40S (small) subunit and 28S and 5.8S rRNAs of the 60S (large) subunit

RNA polymerase III transcribes the 5S rRNA → 60S subunit

40S + 60S join during protein synthesis to form the whole 80S ribosome

prokaryotic ribosomal subunits

50S and 30S large and small subunits create the complete 70S ribosome

initiation

beginning of translation

elongation

continuation of translation; three-step cycle that is repeated for each amino acid added to the protein after the initiator, methionine; moves in the 5′ to 3′ direction, amino (N-) to carboxyl (C-) terminus

termination

end of translation; hydrolyses polypeptide chain, released from the tRNA in the P site, and the two ribosomal subunits will dissociate

Shine–Dalgarno sequence

small prokaryotic ribosomal subunit binds in the 5’ UTR of mRNA

initiator tRNA

binds to start codon

prokaryotes: N-formylmethionine (fMet)

eukaryotes: methionine

initiation factors (IF)

helps large subunit bind to small subunit, forming the completed initiation complex; not permanently associated with the ribosome

A (aminoacyl) site

holds the incoming aminoacyl-tRNA complex

P (polypeptide) site

holds the tRNA that carries the growing polypeptide chain; also where the first amino acid (methionine) binds because it is starting the polypeptide chain

peptide bond

bond between amino acids; formed as the polypeptide is passed from the tRNA in the P site to the tRNA in the A site; catalysed by peptidyl transferase

peptidyl transferase

an enzyme that is part of the large subunit; catalyses peptide bonds; uses GTP

E site

where the now inactivated (uncharged) tRNA pauses transiently before unbinding and exiting the ribosome

Elongation factors (EF)

assist by locating and recruiting aminoacyl-tRNA along with GTP, while helping to remove GDP once the energy has been used

signal sequences

designate a particular destination for the protein

secretion: ribosome → endoplasmic reticulum (ER) → translated directly into the lumen of the rough ER → Golgi apparatus → vesicle → exocytosis

other pathways: nucleus, lysosomes, cell membrane

release factor (RF)

binds to the termination codon; water molecule added; allows peptidyl transferase and termination factors to hydrolyze the completed polypeptide chain from the final tRNA

chaperones

specialised class of proteins that assist in the protein-folding process

cleavsge event

protein cleaved from a larger, inactive peptide to achieve its active form

ex. insulin, signal sequence

quaternary structure

subunits come together to form the functional protein

ex. hemoglobin

Phosphorylation

addition of a phosphate group (PO₄²⁻) by protein kinases to activate or deactivate proteins; most commonly seen with serine, threonine, and tyrosine

Carboxylation

addition of carboxylic acid groups to proteins, usually to serve as calcium-binding sites

Glycosylation

addition of oligosaccharides as proteins pass through the ER and Golgi apparatus to determine cellular destination

Prenylation

addition of lipid groups to certain membrane-bound enzymes

operon

cluster of genes transcribed as a single mRNA in prokaryotes

Jacob–Monod model

used to describe the structure and function of operons; contain structural genes, an operator site, a promoter site, and a regulator gene

structural gene

codes for the protein of interest

operator site

nontranscribable region of DNA that is capable of binding a repressor protein; Upstream of the structural gene

promoter site

provides a place for RNA polymerase to bind; further upstream than operator site

regulator gene

codes for a protein known as the repressor, futher upstream than promoter

inducible systems

the repressor is bonded tightly to the operator system and thereby acts as a roadblock; an inducer must bind the repressor protein so that RNA polymerase can move down the gene

negative control mechanisms

the binding of a protein reduces transcriptional activity

lac operon

codes for lactase and lactose-specific transport proteins; induced by the presence of lactose; most efficient when lactose is high and glucose is low

catabolite activator protein (CAP)

transcriptional activator used by E. coli when glucose levels are low; Falling levels of glucose → increase in cAMP → binds to CAP → conformational change → bind the promoter region of the operon → increasing transcription of the lactase gene

positive control mechanisms

the binding of a molecule increases transcription of a gene

Repressible systems

allow constant production of a protein product; repressor made by the regulator gene is inactive until it binds to a corepressor; often engative feedback

trp operon

tryptophan corepressor; stops production of tryptophan in cell if too much made/in local environment

Transcription factors

transcription-activating proteins that search the DNA looking for specific DNA-binding motifs

DNA-binding domain

binds to a specific nucleotide sequence in the promoter region or to a DNA response element to help in the recruitment of transcriptional machinery

response element

a sequence of DNA that binds only to specific transcription factors

activation domain

allows for the binding of several transcription factors and other important regulatory proteins, such as RNA polymerase and histone acetylases, which function in the remodeling of the chromatin structure

amplified

increased

enhancer

Several response elements grouped together; allows for the control of one gene’s expression by multiple signals; up to 1000 base pairs away from the gene they regulate and can even be located within an intron

ex. cyclic AMP (cAMP) and cyclic AMP response element-binding protein (CREB); cortisol and glucocorticoid (cortisol) receptor; estrogen and estrogen receptor

Gene Duplication

increase the expression of a gene product; duplicated on same chromosome or in parallel (repeated replication)

Heterochromatin

tightly coiled DNA that appears dark under the microscope; its tight coiling makes it inaccessible to the transcription machinery, so these genes are inactive

Euchromatin

looser and appears light under the microscope; the transcription machinery can access the genes of interest, so these genes are active

histone acetylases

acetylate lysine residues found in the amino terminal tail regions of histone proteins