RNA and Proteins Flashcards
Gene
– a segment of DNA that contains all the information necessary for the synthesis of a function product (protein or RNA)
o Composed of:
untranscribed promotor and enhancer regions that are necessary to regulate transcription
transcribed coding regions (exons) that are translated into protein
transcribed non-coding (introns) which are removed post transcriptionally
transcribed untranslated regions (UTR) at the 5’ and 3’ ends that regulate translation and mRNA stability
Transcription Initiation
o RNA polymerase does not need a primer to provide first 3’OH; it binds to DNA weakly and non-specifically
o 2 type of transcription factors that help RNA polymerase begin to transcribe in the proper location and at correct rate to make required amount of mRNA
Basal/general transcription factors
Transcriptional activators (transcription factors, TSFs)
Basal/general transcription factors
• Bind as multi-protein complex to TATA box and interacts with RNA pol.
• Same complex for every gene
• Regulates low, basal level transcription
• Not regulated in response to environment
TATA Binding Protein (TBP) – 1st transcription factor to bind to initiation complex
• Part of TFIID that binds directly to TATA
• Completion of initiator complex causes conformational change
• Kinks DNA to separate strands
TFIIH
• Last basal transcription factor to bind to initiation complex
• Multi-subunit protein with helicase activity that uses ATP to unwind helix and separate DNA strands
• Kinase activity phosphorylates the C terminal domain of RNA polymerase to signal transcription to begin
• Also plays roll in transcription-coupled repair of DNA
Transcriptional Activators
Bind to enhancer elements in regulatory regions of genes and influence rate of transcription initiation
• Recruit histone acetyl transferases and/or chromatin remodeling complexes
o Acetylation leads to euchromatin formation
o Deacetylation leads to heterochromatin formation
• AND/OR have direct interactions with general transcription factors, or indirect interactions through mediator proteins
Each factor binds to a certain nucleotide sequence in DNA (consensus site; enhancer element) via its DNA binding domain
• Consensus binding sequences – binding site of a given transcription factor can be slightly different on different genes – purines/pyrimidines may vary
o Varying binding affinity
• Each gene is bound by a different set of activators
Regulate transcription in response to environmental signals
Facilitate the formation of basal initiation complex
Repressors
– can interfere with the activity of transcriptional activators in multiple ways
o Competition for same binding site as activator
o Binding to and obstructing the activation domain of activator
o Recruiting histone deacetylases or other proteins to cause heterochromatin formation
Transcriptional Elongation
o One strand (coding strand) is held out of way while the other strand (template strand) is copied
o 1st nucleotide remains a triphosphate and is protected by adding a 5’ cap (= 7 methylguanosine)
o RNA sequence is complementary to the template strand except with U’s replacing T’s
o 3 major RNA polymerases
o DNA will re-anneal back together after transcription
5’ Cap
– 7 methylguanylate – occurs very quickly
Backward 5’ to 5’ linkage
1st 1 or 2 ribonucleotides are also methylated
Added by a capping enzyme (guanyl transferase) that associated with the phosphorylated CTD of RNA polymerase II
Provides mRNA stability
Aids in transport of mature mRNA from nucleus to cytoplasm
Necessary for efficient translational initiation
post-transcriptional processing of mRNA
RNA polymerase II carries some pre-mRNA processing proteins on its tail (the phosphorylated C-terminal domain – CTD); the proteins are transferred to newly transcribed mRNA when necessary
• Some DNA repair enzymes also travel with RNA polymerase in this way
splicing
Introns are intervening sequences that do not encode proteins; they are removed in the nucleus and the exons are spliced together before mature mRNA is transported out
• Involves small nuclear RNAs (snRNAs) which are associated with proteins to form small ribonucleoprotein particles (snRNPs)
• Some snRNPs recognize consensus sequences at exon/intron and intron/exon junctions
Alternative splicing – some mRNAs can be spliced in different ways to yield different proteins from the same pre-mRNA transcript; ~60% of genes
~15% of all genetic diseases result from mutations that affect mRNA splicing
polyaadenylation
mRNA is cleaved downstream of polyA signal (AAUAAA)
polyA polymerase adds 100-200 A’s to the 3’ end; then polyA binding protein binds
nuclease binds the 5’ end and degrades the trailing RNA; when it reaches the RNA polymerase, transcription is terminated
poly A is added in the nucleus but sometimes lengthened in the cytosol
provides mRNA stability
involved in transport of mature mRNA from nucleus to cytosol
Contributes to efficient translational initiation
tRNA structure
– cloverleaf structure – 70-90 nt long – 31 kinds
o 3’ end – attaches amino acid
o Anticodon
o D and T loops
tRNA processing
o 16 nucleotide sequence at 5’ end is cleaved by RNase P
o 14 nucleotide intron in the anticodon loop is removed
o Uracil residues at 3’ end are replaced by CCA sequence – found in all mature tRNAs
o Many bases are converted to characteristic modified bases
ribosomal RNA
o Single gene encodes large 45S precursor
o 250-300 copies of the gene clustered together
o Some nucleotides of rRNA precursor are chemically modified
o Processed by cleavage rRNA is responsible for most of the catalytic activity of ribosome
o Ribosomal subunits are assembled in nucleolus by ribosomal proteins
miRNA
– estimated to be ~1000
o may be involved in regulation of 2/3 of genes
o transcribed by RNA polymerase II
some processed from introns or 3’UTR of pre-mRNA
most processed from pri-miRNAs – transcripts of 100-1000 nucleotides
o miRNA acts as a “guide sequence” that brings nuclease into contact with targets mRNAs
o if sequence is complimentary to miRNA, argonaute slices the mRNA, removing the polyA tail and making the mRNA vulnerable to nucleases
o if sequence is not fully complimentary, no splicing occurs but translation is inhibited and the mRNA is destabilized by moving it to P-bodies (processing bodies) in the cytosol where it is eventually degraded
processing of miRNA
o miRNA sequence folds to form hairpin of ~70 nucleotides with imperfect base pairing in stem
o an RNase (Drosha) cleaves hairpin to produce pre-miRNA
o pre-miRNA goes to cytosol
o dicer RNase process the pre-miRNA to produce a double stranded miRNA that’s 21-23 nt long
o one strand binds with an argonaute protein to form a mature RISC (RNA-induced silencing complex)
Genetic Code
– 3 mRNA nucleotides form a codon which encodes an amino acid o Non-overlapping, no punctuation – starts at AUG (codes for methionine) until an in-frame stop codon is reached (UAA, UAG, UGG) o Specific – given code always encodes the same amino acid o Degenerate/redundant – given amino acid may be encoded by more than 1 codon Due to “wobble” in the binding between tRNA and mRNA codon • 5’ base of tRNA anticodon undergoes more movement than the other 2 o Amino (N) terminus of protein corresponds to the 5’ end of mRNA o Carboxy C terminus of protein corresponds to the 3’ end of mRNA
Types of Point Mutations
o Frameshift – caused by insertion or deletion of any number of nucleotides not divisible by 3
o Missense – new codon encodes different amino acid
o Nonsense – new codon is a stop codon resulting in premature termination
o Silent – new codon encodes the same amino acid; change in DNA sequence but not protein
Activation or Charging of tRNAs
– aminoacyl tRNA synthetases
o Family of 20 enzymes
o Each recognizes one amino acid
o 2 active sites – 1 for amino acid and 1 for tRNA
o “double sieve” proofreading – makes sure correct amino acid is added to tRNA
Synthesis site - Rejects amino acids that are larger than correct one
Editing Site – rejects amino acids that are smaller than the correct one
Initiation of Protein Synthesis - characteristics
Involves eukaryotic initiation factors (eIFs)
• eIF-2-GTP has to be recycled – regulation point
involves special initiator tRNA charged with methionine
involves recognition of 5’ cap and polyA tail by eIF’s
use energy from ATP and GTP
eIF-2-GDP must be reactivated to eIF-2-GTP before it can participate in more initiation
• allows for global regulation of translation under certain conditions
initiation of protein synthesis mechanism
o initiation begins begins by attaching methionine amino acid to a specific initiator tRNA
o initiator met-tRNA forms comples with eIF-2-GTP
other eIFs separate the ribosomal subunits and bind the small 40S subunit
o initiator tRNA complex binds to the small ribosomal unit to form an initiator complex
o mRNA is bound by initiation factors, including some that recognize the 5’ cap and polyA tail
o initiator complex binds to mRNA and uses energy from ATP to move along the mRNA until it find the AUG codon; complementary base pairs are formed between start codon and anticodon of tRNA; tRNA for methionine goes directly into P site
o 60S subunit is added in a reaction that hydrolyses GTP and releases initiation factors
Elongation of Peptide Chain
The initiator met-tRNA complex occupies the P site (the “peptidyl” or donor site)
o A tRNA containing an anticodon complementary to the next codon and charged with the correct amino acid enters the A site (the “acceptor”, “aminoacyl” or entry site).
o If the hydrogen bonding between the codon and the anticodon is correct, GTP is hydrolyzed and the elongation factor is released along with GDP.
o Peptidyl transferase is an enzymatic activity associated with an rRNA in the large 60S ribosomal subunit. It transfers the peptide chain from the tRNA in the P site to the amino group of the amino acid attached to the tRNA at the A site to form a peptide bond. This is a condensation reaction
o Peptide bond formation is accompanied by conformational changes in the large ribosomal subunit that result in shifting the two tRNAs to the E and P sites.
o Another series of conformational changes moves the mRNA exactly three nucleotides through the ribosome and resets the ribosome so the A site is open
o Elongation proceeds as a repetition of these reactions, adding one amino acid at a time to the growing peptide chain.
Termination of Protein Synthesis
o When a termination codon is in the A site, eRF-GTP (release factor) binds in the A site
o Protein is released from tRNA by peptidyl transferase in a reaction that involves addition of water to the COO- group of the last amino acid and the hydrolysis of GTP
o Uncharged tRNA dissociated from ribososme and the ribososme dissociates from mRNA
polyribosomes
• More than one ribosome can translate the same mRNA at the same time (polyribososmes)
o eIF that interacts with both 5’ and 3’ end of mRNA transcript and forms a circle so that the ribosome can restart another translation as soon as it finishes a previous one
