RNA And Protein Synthesis Flashcards
Ribosomal RNA
. 4 of them (28S, 18S, 5.8S, and 5S) . Assoc. w/ proteins to form ribosomes . Required for protein synthesis . Accounts for 80% total RNA in cell . Non-coding (functional)
Transfer RNA
. Carry AA . 1 specific tRNA for each AA . Required for protein synthesis . Make up 15% total RNA . Non-coding (functional)
Messenger RNA
. Carry genetic info encoded by DN from nucleus to cytosol
. Transcripts (protein coding)
. Heterogenous (size and sequence differs)
. 5% total RNA
Long non-coding RNA (lncRNA)
. Antisense: transcribed from sense strand of gene nucleotide sequence, not processed/no splicing, bind mRNAs transcribed from antisense strand to repress gene expression
. Sense: transcribed and processed like primary transcripts of protein-coding genes, regulate neighboring genes
. Non-coding (functional)
Micro RNA (miRNA)
. 22 nucleotides in length
. Bind to 3’ UTR of specific mRNAs
. Regulate gene expression by repressing protein production/triggers mRNA degradation
. Non-coding (functional)
MiRNAs in human disease
. RNA interference (gene regulation via miRNA)
. Critical for normal development
. 50% protein-coding genes regulates by this
. A;teres patterns of miRNA shown to be assoc. w/ various diseases and cancers
Protein-coding gene
. Genes w/ nucleotide sequence that is transcribed and is translated to produce protein
. Divided into coding and regulatory region/transcriptional start site
. Coding region divided into coding Exons and non-coding introns
Consensus sequences
. Recognition markers/landmarks for protein binding
. DNA sequences highly conserved and found in noncoding regions
. Regions that modulate gene expression
. Bound by transcription factors or other regulatory proteins
. Ex: TATA box
Promoter sequence
. Region that selects or determines start site of RNA synthesis
. Contains TATAbox 15-30 bps upstream from transcription start site
Binding of transcription factors to TATA box facilitates recruitment of _____
RNA polymerase II
Splice acceptor and donor sequences
. Consensus sequences found at 5’ and 3’ ends of introns
. Needed for removal of introns out of primary transcript (splicing)
. Introns start w/ splice donor site (always GU, GT in DNA) and end w/ splice acceptor site (Ends w/ AG)
Transcription
. Production of RNA
. Occurs at nucleus
. only 1 of 2 strands is used as template (template strand)
. Catalyzed by RNA polymerase that read DNA in 3’-5’ direction to produce complementary RNA, have proofreading activity
RNA polymerase I
Synthesized precursors of rRNA in nucleolus
RNA polymerase II
. Produces mRNAs and miRNAs in nucleus
RNA polymerase III
. Catalyzes synthesis of tRNAs and 5S rRNAs in nucleus
Steps in production of mRNA by RNA polymerase II (RNAP)
. Initiation: chromatin remodeling (to open), assembly of preinitiation complex and recruitment of RNAP
. Elongation: local unwinding of DNA, RNAP moves along template strands catalyzes creation of transcript that grows in 5’-3’
. Termination: RNAP encounters stop signal, enzyme and transcript released
Preinitiation complex
. 6 general transcription factors (GTFs)
. Each GTf is a multiprotein complex
. Proper assembly required for proper recruitment and correct positioning of RNAP
Typical structure of mRNA
. Produced in cytosol
. Have exons and introns
. When initially produced, contain regulatory sequences at 5’ and 3’ ends called untranslated regions (needed for trailing and translational efficiency) and are not part of product
Regulatory features of mRNA transcription through RNAP II
. Region upstream of start site are regulatory
. Basal promoters, enhancers/repressors, and response elements
Basal Promoters
. Ensures basal expression
. Found adjacent to transcriptional start site
. TATA box: prox. Element, specifics start site by directing RNAP to correct site, ensures fidelity of initiation
. CAAT and GC boxes: protein-DNA interactions determine frequency of transcription initiation
Enhancers/repressors, insulators, and response elements
. Participate in regulated expression
. Found far away from start site
. Enhance/repress expression
. Mediate response to signals/hormones by inc/dec. rate of initiation
. Function in orientation-independent manner
RNA transcription w/ introns is called ____
. Primary transcript
. Pre-mRNA
. Heterogenous nuclear RNA (hnRNA)
.
How pre-mRNA is processed to get to cytosol
. Addition of 5’ cap (7-methylguanosine triphosphate) to 5’ end of transcript
. Addition of poly A tail: signaled by polyadenylation signal (AAUAAA) at 3’ end of primary transcript
. Splicing of introns: removed, needs spliceosome
Spliceosome
. Combo of pre-mRNA and complex of 5 small nuclear RNAs (U1, 2, 5, and 4/6) and over 50 proteins (snRNPS)
Branch site in intron splicing
. Hydroxyl group of adenosine in intron required for splicing
Exonic splicing enhancers and silencers (ESE and ESS)
. Influence constitutive and alternative splicing
. Commonly altered in human disease
Steps of intron splicing
. 1: spliceosome recognizes splice donor/acceptor site
. 2: branch site OH attacks 5’-phosphate of splice donor at 5’ end of intron to form 2’-5’ phosphodiester forming lariat when cleaved
. 3: 3’-OH of upstream exon attacks splice acceptor at 5’-P of downstream exon, intron removed as phosphodiester bond forms to join exons
. Exported into cytoplasm through nuclear pores
Rifampin uses and mechanism
. Antibiotic used to treat TB
. Prevents bacterial cell growth by inhibiting RNA synthesis
. Targets bacterial DNA-dependent RNA polymerase enzyme
. Binds to beta-subunit of bacterial RNAP to block transcription initiation
Dactinomycin (Actinomycin D)
. Antibiotic/anti-neoplasticism agent used to treat Wilm’s tumor and sarcomeres . Prevents bacterial growth by inhibiting RNAP movement and RNA elongation during DNA-primed RNA synthesis by binding to DNA template . Member of actinomycine class of antibiotics isolated from soil bacteria of Strep
Alpha-amanitin
. Toxin in mushrooms
. Inhibits human RNAP II movement along DNA template to prevent RNA synthesis
. Symptoms after 24 hrs (diarrhea and cramps) then liver and kidney damage preceding total system failure and death w/in 1 week
Codons
. Sequence of 3 nucleotides for specific amino acid
. Written and read in 5’-3’ direction
. 64 different ones
. 61 of 64 code from 20 common AA
. 3 are termination signals (UAG, UGA, and UAA)
Characteristics fo genetic code
. Specific: codon always codes for same AA
. Universal: genetic code conserved across species
. Degenerate/redundant: AA may be specified by more than one codon
. Nonoverlapping and commaless: code read continuously from fixed point, sequence of bases processed 3 at a time
Components required for translation
. MRNA: template
. AA: must be available to put in final product
. TRNA: translators of genetic code
. Ribosomes: rRNA and protein complex
. Protein factors: specific initiation, elongation, and termination factors required
. Energy sources: ATP and GTP
TRNA structure
. AA attachment site at 3’ end
. Anticodon, 3-base recognition requests that allows complementary binding to correct series of 3 bases on mRNA
Aminoacyl-tRNA synthetases
. Highly selective enzymes
. Needed for attachment of AA to corresponding tRNAs to form aminoacyltRNAs
. Uses ATP to catalyze 2-step rxn resulting in covalent attachment of carboxyl group of AA to 3’ end of tRNA
Ribosome mechanism
. 60S subunit catalyzes formation of peptide bonds btw AA
. 40S subunit binds mRNA, responsible for accuracy of translation
. A, P, and E binding sites for tRNA
. A: binds incoming aminoacyl-tRNA, specifies next AA added to polypeptide chain
. P: peptidyl-tRNA carries growing chain
. E: empty tRNA that is about to exit ribosome
How tRNA is attached to it’s specified AA
. Covalently attachment
. Charged when bound to AA, uncharged when not bound to AA
. When AA is attached to tRNA it is activated
Post-translational modification of protein types
. Co-translational or post-translational
Proteolytic cleavage (trimming)
. Most common
. Proteolysis removal of their leading Met residue after it emerges from ribosome
. If proteins synthesized as inactive precursors, they are activate by limited proteolysis (trypsinogen) or removal of polypeptides (proinsulin to insulin)
Covalent posttranslational modifications
. Modified by covalent attachment to chemical groups, sugars, lipids, or small proteins to specific AA
Hydroxylation posttranslational modifications
. Pro and Lys residues of collagen alpha-chains hydroxylated in ER
. Requires Vit. C
Phosphorylation as posttranslational modification
. Hydroxyl groups of Ser and Thr and sometimes TYr residues
. Catalyzed by protein kinases, reversed by phosphatases
. Activates or inhibits function
Glycosylation as posttranslational modification
. Attachment of carb chains to hydroxyl groups of Ser or Thr (O-linked) or Asn (N-linked)
. Normally secreted from cell or become part of plasma membrane
What does the 5’ cap of mRNA do?
. Prevents degradation by exonuclease
. Facilitates binding of mRNA to ribosomes during protein synthesis
How is it added and What does addition of poly(A) tail to mRNA do?
. Signal recognized by endonuclease that cleaves RNA about 20 nucleases
. New 3’ end serves as primer for enzymatic addition of 250 adenine ribonucleotides
. Important for nuclear export, translation, and mRNA stability
Premature termination codons
. splicing mutation creates truncated protein w/ impaired function
Nonsense mediated decay
. Doesn’t act on single exon genes, depends on RNA splicing
. Exon-junction complexes bind 20 nt upstream of 3’ end of transcribed exons and remain bound in mature mRNA
. Ribosome removes them in translation one-by-one as protein synthesis progresses
. If PTC detected, it is released from ribosome w/ EJCs bound to signal degradation
Where will fame-shift PTC occur and escape NMD most likely?
55 nt upstream of last exon
Sonic hedgehog molecule
. Secreted signaling molecule
. Critical role during CNS development, face, limbs, and other body parts
. Expressed and secreted to form morphogenetic gradient that influences tissue patterning
. Coding sequence mutations are a cause of holoprosencephaly (HPE)
. Zone of polarizing activity in vertebrate limb importance
. Specific expreassign of SHH here is critical to having normal limbs
. ZPA Regulatory sequence (ZRS): long-distance enhancer element identified as direct SHH expression to ZPA
ZRS enhancer sequence is found ____
. 1st intron of neighboring LMBR1 gene
. 1 Mb away from SHH
. Mutations cause polydactyly
ZRS enhancer point mutations cause ___
Preaxial polydactyly (double thumb)
Position effects
Chromosomal rearrangements caused by repositioning of long range enhancers required for normal gene expression
. Creates mutations like the thalassemias
Imitating codon for translation
AUG, codes for methionine
Rate limiting step of translation
. Binding of methylguanosine cap binding complex to the 5’ cap of mRNA
elongation in translation steps
. Binding of aminoacyl-tRNA to the A site (eukaryotic elongation factors assist in delivery of aminoacyl-tRNA whose codon appears next)
. Peptide bond formation: alpha-amino group of new aminoacyl-tRNA in A site carries nucleophilic attack on esterified carboxyl group of peptidyl-tRNA occupying P site
. Rxn catalyzed by peptides-transferase activity of 60S ribosomal subunit
. Growing peptide chain now attached to tRNA in A site
Translocation steps in translation
. Peptide moiety is removed from tRNA in P site
. TRNA dissociates from P site and moves into E site
. Elongation factors tranlocate the newly formed peptidyl-tRNA into empty P site and A site becomes free
How primary miRNAs (pri-miRNAs) transcribed from genome?
. Via RNA Pol II or III
. Shorter inverted sequences that form double stranded RNA stem loop structure
. Processed into pre-miRNAs stemloop in nucleus by microprocessor complex comprised of DGCR8 and drosha (ribonuclease)
How pre-miRNAs is modified before translation?
. Pre-miRNA exported into cytoplasm via exportin 5/Ran GTP complex
. Further processed into mature miRNA by Dicer
. 1 of 2 strands of mature miRNA is loaded into 1 of 4 members of argonaute family to form miRISC complex
How miRNA is translated
. Chosen strand is guide miRNA
. MiRISC complex binds mRNAs at miRNA response elements (MREs)
. If guide miRNA contains mismatch in nucleotide sequence, gene expression may be inhibited through mechanisms leading to mRNA degradation or translational repression
. If guide RNA in miRISC complex is perfect match to an mRNA’s MRE, then RNA endonuclease activity cleaves mRNA
