Review from last time • Coding and non-coding repeats make up the moderately repetitive portion of the eukaryotic genome • Polyploidization and gene duplication contribute to structural and functional genome change • Transposable elements contribute to structural and functional genome change • Comparative genomics is a useful technique to determine the conserved (likely functional) portions of a genome
70
Embed
Review from last time Coding and non-coding repeats make up the moderately repetitive portion of the eukaryotic genome Polyploidization and gene duplication.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Review from last time
• Coding and non-coding repeats make up the moderately repetitive portion of the eukaryotic genome
• Polyploidization and gene duplication contribute to structural and functional genome change
• Transposable elements contribute to structural and functional genome change
• Comparative genomics is a useful technique to determine the conserved (likely functional) portions of a genome
Chapter 11:Gene Expression:
From Transcription to Translation
This Chapter in One Slide
details
details
details
deta
ils
details details
detailsdetails
details
details
details
details
details
details
detailsdetails
deta
ils
det
ails
details
Gene Expression• RNA – Ribonucleic acid
– Slightly different from DNA– Uracil instead of Thymine
• RNA is critical to all gene expression• mRNA – messenger RNA; created from a
DNA template during transcription• tRNA – transfer RNA; carriers of amino
acids; utilized during translation• rRNA – ribosomal RNA; the site of translation• Other RNAs – snoRNA, snRNA, miRNA,
siRNA• Many RNAs fold into complex secondary
structures
Transcription• Transcription – the process of copying a DNA template
into an RNA strand• Accomplished via DNA dependent RNA polymerase (aka
RNA polymerase)
Transcription• By the end of this series, you should be able to explain
• Most genes are transcribed simultaneously by numerous polymerases
• Polymerase moves along DNA in 3' —> 5' direction• Complementary RNA constructed in ____ direction
• RNAn + NPPP —> RNAn+1 + PPi
Transcription• How does the polymerase know where to start?• Promoter = the assembly point for the transcription
complex• RNA polymerases cannot recognize promoters on their
own - transcription factors• Transcription factors - enzymes have evolved to
recognize (physically interact with) specific DNA sequences and with other proteins
• The promoter is one such DNA sequence
Transcription• Prokaryotic Transcription• Similar DNA sequences are seen associated with genes in roughly
the same location for multiple genes in bacteria– The consensus sequence is the most common version of such a
conserved DNA sequence
– DNA sequences just upstream from a large number of bacterial genes have 2 short stretches of DNA that are similar from one gene to another (-35 region & -10 region)
Transcription• Prokaryotic Transcription• Bacterial promoters are located just upstream of the
RNA synthesis initiation site– The nucleotide at which transcription is initiated is called +1; the
preceding nucleotide is –1– DNA preceding initiation site (toward template 3' end) are said to
be upstream– DNA succeeding initiation site (toward template 5' end) are said
to be downstream
• Prokaryotic Transcription• One RNA polymerase with 5
subunits tightly associated to form core enzyme
• Core enzyme minus sigma (σ) factor will bind to any DNA.– By adding σ, RNA pol will bind
specifically to promoters (-10 & -35 sequences)
Transcription
Transcription• Eukaryotic vs. Prokaryotic Transcription• Much of what we know is derived from
studies of RNA pol II from yeast– 1. Seven more subunits than its bacterial
RNA pol– 2. The core structure & the basic
mechanism of transcription are virtually identical
– 3. Additional subunits of eukaryotic polymerases are thought to play roles in the interaction with other proteins
– 4. Eukaryotes require a large variety of accessory proteins or transcription factors (TFs)
Review from last time
• Basic ideas behind transcription and translation• To get from DNA to functional protein, many types of
RNA are critical• RNA differs chemically from DNA in only two ways• RNA tends to form secondary structures• RNA polymerase initiates transcription at promoter sites
with the aid of transcription factors like sigma (in prokaryotes)
• Promoters are DNA sequences that act to direct RNA polymerases to the appropriate position
Transcription• Eukaryotic Transcription - one major difference• Three distinct RNA polymerases, each responsible for
synthesizing a different group of RNAs– RNA polymerase I (RNA pol I) - synthesizes the larger rRNAs
(28S, 18S, 5.8S)– RNA polymerase II (RNA pol II)- synthesizes mRNAs & most
small nuclear RNAs (snRNAs & snoRNAs)– RNA polymerase III (RNA pol III) - synthesizes various small
RNAs (tRNAs, 5S rRNA & U6 snRNA)
Transcription• Eukaryotic Transcription - RNA processing• All major RNA types (mRNA, tRNA, rRNA) must be processed
– Terminology– The primary (1°) transcript is equivalent in length to the DNA
transcribed– The corresponding segment of DNA from which 1° transcript is
transcribed is called transcription unit– The 1° transcript is short-lived; it is processed into smaller,
functional RNAs– Processing requires variety of small RNAs (90 – 300 nucleotides
long) & their associated proteins
Transcription – mRNA• Messenger RNAs (mRNA)• Transcribed by RNA pol II• Remember this?• http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf
• Polymerase II promoters lie to 5' side of each transcription unit– In most cases, between 24 & 32 bases upstream from
transcription initiation site is a critical site– Consensus sequence that is either identical or very similar to
5'-TATAAA-3‘, the TATA box– The site of assembly of a preinitiation complex
• contains the GTFs & the polymerase• must assemble before transcription can be initiated
Transcription – mRNA• The preinitiation complex• Step 1 - binding of TATA-binding
protein (TBP)– Purified eukaryotic polymerase, cannot
recognize a promoter directly & cannot initiate accurate transcription on its own
– TBP is part of a much larger protein complex called TFIID
– TBP kinks DNA and unwinds ~1/3 turn
Transcription – mRNA• The preinitiation complex• Step 2 – Binding of ~8 TAFs (TBP-
associated factors) to make up the complete TFIID complex
• Step 3 – Binding of TFIIA (stabilizes TBP-DNA interaction) and TFIIB (involved in recruiting other TFs and RNA pol II)
Transcription – mRNA• The preinitiation complex• Step 4 – RNA pol II and TFIIF bind via
recruitment by TFIIB• Step 5 – TFIIE and TFIIH bind• TFIIH is the key to activating
transcription in most cases• TFIIH is a protein kinase –
phosphorylates proteins• TFIIH may also act as a helicase
Transcription – mRNA• The preinitiation complex• All these general transcription factors and pol II are enough to
generate basal transcription• Transcription can be upregulated or downregulated by a huge
diversity of other cis and trans acting factors to be discussed in chapter 12.
• Once an mRNA is produced, it must be processed.• Processing involves the addition of a cap, the addition of a poly-A
tail, and splicing out of introns.
RNA processing – mRNA• Transcription generates
messenger RNA– A continuous sequence of nucleotides
encoding a polypeptide– Transported to cytoplasm from the
nucleus– Attached to ribosomes for translation– Are processed to remove noncoding
segments– Are modified to protect from
degradation and regulate polypeptide production
RNA processing – mRNA• RNA polymerase II assembles a 1° transcript that is
complementary to the DNA of the entire transcription unit• 1° transcript contains both coding (specify amino acids)
and noncoding sequences
• Subject to rapid degradation in its raw state
RNA processing – mRNA• 5’ cap• 5' methylguanosine cap forms very soon
after RNA synthesis begins– 1. The last of the three phosphates is
removed by an enzyme– 2. GMP is added in inverted
orientation so guanosine 5' end faces 5' end of RNA chain
– 3. Guanosine is methylated at position 7 on guanine base while nucleotide on triphosphate bridge internal side is methylated at ribose 2' position (methylguanosine cap)
RNA processing – mRNA• 5’ cap• Possible/known functions of 5’
cap– May prevent exonuclease digestion
of mRNA 5' end, – Aids in transport of mRNA out of
nucleus – Important role in initiation of mRNA
translation
RNA processing – mRNA• Polyadenlyation• The poly(A) tail – 3' end of most mRNAs contain a string
of adenosine residues (100-250) that forms a tail– Protects the mRNA from degradation– AAUAAA signal ~20 nt upstream from poly(A) addition site– Poly(A) polymerase, poly(A) binding proteins, and several
cleavage factors are involved– http://www.as.wvu.edu/~dray/219files/mRNAProcessingAdvanced.wmv
• U2 is recruited by proteins associated with an exon splice enhancer (ESE) within the exon
RNA processing – mRNA• mRNA processing –
Splicing• 2. U2 recruits U4/U5/U6
trimer• U6 replaces U1, U1 and U4
released• U5 binds to upstream exon
RNA processing – mRNA• mRNA processing –
Splicing• 3. U6 catalyzes two
important reactions– Cleavage of upstream exon
from intron (bound to U5)– Lariat formation with A bulge
on intron
• Exons are ligated• U2/U5/U6 remain with intron
RNA processing – mRNA• mRNA processing – Splicing• Several lines of evidence suggest that it is the RNA in
the snRNP that actually catalyzes the splicing reactions– 1. Pre-mRNAs are spliced by the same pair of chemical
reactions that occur as group II (self-splicing) introns– 2. The snRNAs needed for splicing pre-mRNAs closely
resemble parts of the group II introns
• Proteins likely serve supplemental functions– 1. Maintaining the proper 3D structure of the snRNA– 2. Driving changes in snRNA conformation– 3. Transporting spliced mRNAs to the nuclear envelope– 4. Selecting the splice sites to be used during the processing of
a particular pre-mRNA
RNA processing – mRNA• mRNA processing –
Splicing• Group II intron self-splicing
summary (rare)
RNA processing – mRNA• Implications of RNA catalysis and splicing• The RNA world
– Which came first, DNA or protein?... Apparently, it could have been RNA– Information coding AND catalyzing ability
• Alternative splicing– Allows one gene to encode multiple protein products
• Intron sequences actually encode some snoRNAs• Evolutionary innovation
– Exon shuffling
RNA processing - rRNA• Eukaryotic ribosomes have four
distinct rRNAs: – Three rRNAs in the large subunit
(28S, 5.8S, 5S in humans); – One in the small (18S in humans)
charging of tRNAs with amino acids• ~20 different versions for the 20 different aa’s
Translation• Ribosome structure• Each ribosome has 3 sites
for association with tRNAs; the sites receive each tRNA in successive steps of elongation cycle– A (aminoacyl) site -– P (peptidyl) site – E (exit) site -
• A channel for the nascent polypeptide to exit is also present
Translation• Ribosome structure• tRNAs bind within these sites & span the gap between
the 2 ribosomal subunits– The anticodon ends of the bound tRNAs contact the small
subunit, which plays a key role in decoding the information contained in the mRNA
– The amino acid-carrying ends of bound tRNAs contact the large subunit, which plays a key role in catalyzing peptide bond formation
Review from last time• 5S rRNA and tRNAs are transcribed by RNA pol III• RNA pol III is unique in its use of an internal promoter• siRNAs and miRNAs are RNAs involved in shutting down
a gene’s function without affecting the gene itself• The genetic code is degenerate• tRNAs are short RNAs that bridge the gap between
information in the mRNA and amino acid chain• The structure of a ribosome is such that three sites are
formed, A, P, and E• As mRNA threads through the ribosome the information
encoded is translated to form an amino acid chain
Translation• Initiation of translation• Step 1. Bind the initiation codon
(AUG, met) to the small ribosomal subunit
• In bacteria• The Shine-Dalgarno sequence on
mRNA is complementary to 16S rRNA• Initiation Factors
– IF1 – attaches 30S subunit to mRNA– IF2 – required for attachment of first tRNA– IF3 – likely prevents bind of large subunit
Translation• Initiation of translation• Step 2. Bind the first tRNA (tRNAMet)• Enters the P site with the help of IF 2
Translation• Initiation of translation• Step 3. Bind the large subunit• IFs released and large subunit binds
Translation• Initiation of translation• Bind the initiation codon (AUG, met) to the small ribosomal subunit• In eukaryotes• Three IFs + tRNAMet bind to 40S subunit• Separately mRNA binds to additiona IFs and PABP• These components come together and scan along mRNA until AUG is
reached• Large subunit binds
Translation• Elongation• The players – EF-Tu/GTP/tRNA
complex– EF-Tu – escorts the tRNA to the
A site– GTP – provides energy– The tRNA - duh
• Any tRNA can enter but only the correct one will induce the conformational changes to induce binding to mRNA
• Once in, GTP -> GDP and Tu-GDP is released
Translation• Elongation• Peptide bond is formed
between aa’s• Peptidyl transferase – a
ribozyme• tRNA in P site is now
uncharged
Translation• Elongation• Translocation of the ribosome
along the mRNA (3 nt)• tRNAs rachet positions• EF-G induced• GTP -> GDP + P required
Translation• Elongation• Release of the uncharged
tRNA and repeat the whole cycle
• ~1/20 second
Translation• Termination• Three codons (UAA, UGA, UAG) have no
complementary tRNAs• Protein released when one is reached• Release factors are required• Bacteria RF1, RF2, RF3• Eukaryotes eRF1, eRF3• Each recognizes particular stop codon much like a tRNA • RF3/eRF3 binds GTP to energize the release of the
polypeptide and disassemble the ribosome• The complete process (for bacteria) is illustrated using
videos on the class website.
Translation
Prokaryote
Eukaryote
• Polyribosomes
Note the difference – Due to presence/absence ofnuclear membrane