Molecular Biology I BIO316 Lecture 10 PDF

Molecular Biology I BIO316 Lecture 10 Posttranscriptional modifications Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Post-transcriptional processes  Many...

Molecular Biology I BIO316 Lecture 10 Posttranscriptional modifications Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Post-transcriptional processes  Many eukaryotic RNAs undergo extensive processing after transcription.  Different classes of RNA will undergo multiple modifications after transcription process. (1) Messenger RNA processing  Messenger RNA functions as the template for protein synthesis.  It carries genetic information from DNA to a ribosome and helps to assemble amino acids in their correct order.  In prokaryotes, mRNA is transcribed directly from DNA.  In eukaryotes, a pre-mRNA (the primary transcript) is first transcribed from DNA and then processed to yield the mature mRNA. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli (1) Messenger RNA processing Associate Professor of Biochemistry and Molecular Genetics  The structure of mRNA:  In the mature mRNA, each amino acid in a protein is specified by a set of three nucleotides called a codon.  Both prokaryotic and eukaryotic mRNAs contain three primary regions.  The 5’ untranslated region (5’ UTR; called the leader), a sequence of nucleotides at the 5’ end of the mRNA,  It does not encode any of the amino acids of a protein.  In bacterial mRNA, this region contains a consensus sequence called the Shine–Dalgarno sequence.  Its Function:  It serves as the ribosome binding site during translation.  It is found approximately seven nucleotides upstream of the first codon translated into an amino acid (called the start codon).  Dr. Rami Elshazli (1) Messenger RNA processing Associate Professor of Biochemistry and Molecular Genetics  In eukaryotic cells, ribosomes bind to a modified 5’ end of mRNA.  The protein-coding region:  It comprises the codons that specify the amino acid sequence of the protein.  The protein-coding region begins with a start codon and ends with a stop codon.  The last region of mRNA is the 3’ untranslated region (3’ UTR; sometimes called a trailer),  It is a sequence of nucleotides that is at the 3’ end of the mRNA and not translated into protein.  Which region of mRNA contains the Shine–Dalgarno sequence?  5’ untranslated region.  3’ untranslated region.  Protein-coding region.  All three regions.  (1) Messenger RNA processing  In bacterial cells, transcription and translation take place simultaneously.  Once the 3’ end of mRNA undergoes transcription, then ribosomes attach to the Shine–Dalgarno sequence near the 5’ end and begin translation.  Because transcription and translation are coupled, there is little opportunity for the bacterial mRNA to be modified before protein synthesis.  In eukaryotic cells, transcription takes place in the nucleus, whereas translation takes place in the cytoplasm. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  (A) Addition of the 5’ Cap  The first modification of eukaryotic pre-mRNA is the addition at its 5’ end of a structure called a 5’ cap.  This capping consists of the addition of:  An extra modified guanine nucleotide (7-methylguanine) attached to the pre-mRNA by a unique 5’–5’ bond.  Methyl groups added to the 2’ position (2’-OH group) of the sugar in the second and third nucleotides.  Methyl group added to the base (N) on the initial nucleotide.  Capping takes place rapidly after the initiation of transcription and, the 5’ cap functions in the initiation of translation.  Function of 5’ Capping:  Cap-binding proteins recognize the cap and attach to it.  Then, ribosome binds to these proteins and moves downstream along the mRNA until the start codon is reached and translation begins.  The presence of a 5’ cap also increases the stability of Dr. Rami Elshazli mRNA and influences the removal of introns. Associate Professor of Biochemistry and Molecular Genetics  (B) The Addition of the Poly(A) Tail  The second modification to eukaryotic mRNA is the addition of 50 to 250 adenine nucleotides at the 3’ end forming a poly(A) tail.  These nucleotides are not encoded in the DNA but are added after transcription in a process termed polyadenylation.  Processing of the 3’ end of pre-mRNA requires the recognition of specific consensus sequences upstream of the cleavage site.  This consensus sequence AAUAAA is usually before 11 to 30 nucleotides upstream of the cleavage site and determines the point at which cleavage will take place. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics (B) The Addition of the Poly(A) Tail  A sequence rich in uracil nucleotides is typically downstream of the cleavage site.  After cleavage has been completed, adenine nucleotides are added to the new 3’ end, creating the poly(A) tail.  Functions of poly(A):  The poly(A) tail confers stability on many mRNAs, increasing the time during which the mRNA remains intact and available for translation before it is degraded by cellular enzymes.  The poly(A) tail also facilitates attachment of the ribosome to the mRNA. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  (C) RNA Splicing  The third modification of eukaryotic pre-mRNA is the removal of introns by RNA splicing.  This modification takes place in the nucleus, before the RNA moves to the cytoplasm.  Splicing requires the presence of three sequences in the intron.  One end of the intron is referred to as the 5’ splice site, and the other end is the 3’ splice site;  These splice sites possess short consensus sequences. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  (C) RNA Splicing  Most introns in pre-mRNAs begin with GU and end with AG.  The third sequence for splicing is at the branch point, which is an adenine nucleotide that lies from 18 to 40 nucleotides upstream of the 3’ splice site.  Most introns in pre-mRNAs begin with GU and end with AG.  Splicing takes place within a large structure called the spliceosome.  The spliceosome consists of five RNA molecules and almost 300 proteins.  The RNA components associate with proteins to form small nuclear ribonucleoproteins (snRNPs).  Introns contain three consensus sequences critical to  The spliceosome is composed of five snRNPs (U1, U2, splicing: 5’ splice site, 3’ splice site, and branch point. U4, U5, and U6).  Splicing of pre-mRNA takes place within a large complex called the spliceosome, which consists of snRNAs and Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics proteins.  Steps of RNA Splicing  Pre-mRNA is spliced in two distinct steps.  In the first step of splicing, the pre-mRNA is cut at the 5’ splice site.  The 5’ end of the intron attaches to the branch point and the intron folds back on itself, forming a structure called a lariat.  In the second step of splicing, a cut is made at the 3’ splice site.  The 3’ end of exon 1 becomes covalently attached to the 5’ end of exon 2.  The intron is released as a lariat.  The intron becomes linear when the bond breaks and is then degraded by nuclear enzymes.  The mature mRNA consisting of the exons spliced together is Dr. Rami Elshazli exported to the cytoplasm, where it is translated. Associate Professor of Biochemistry and Molecular Genetics  These splicing reactions take place within the spliceosome.  Alternative splicing processes  Many eukaryotic mRNAs undergo alternative splicing processing.  A single pre-mRNA is processed in different ways to produce alternative types of mRNA, resulting in the production of different proteins.  In alternative splicing, the same pre-mRNA can be spliced to yield multiple mRNAs that are translated into different amino acid sequences and thus different proteins. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Messenger RNA processing Associate Professor of Biochemistry and Molecular Genetics  How is a mature mRNA produced?  The promoter encompasses about 100 nucleotides upstream of the transcription start site.  It is not transcribed by RNA polymerase II.  All the nucleotides between the transcription start site and the stop site are transcribed into pre-mRNA, including exons, introns, and a long 3’ end.  The 5’ end of the first exon contains the sequence that encodes the 5’ untranslated region.  The 3’ end of the last exon contains the sequence that encodes the 3’ untranslated region.  The pre-mRNA is then processed to yield a mature mRNA.  The first step is the addition of a cap to the 5’ end of the pre-mRNA.  Then, the 3’ end is cleaved at a site downstream of the AAUAAA consensus sequence in the last exon.  Dr. Rami Elshazli Messenger RNA processing Associate Professor of Biochemistry and Molecular Genetics  Immediately after cleavage, a poly(A) tail is added to the 3’ end.  Finally, the introns are removed to yield the mature mRNA.  The mRNA now contains:  5’ and 3’ untranslated regions, which are not translated into amino acids.  The nucleotides that carry the protein-coding sequences.  (2) Transfer RNA processing  The structure of tRNA:  Transfer RNA serves as a link between the genetic code in mRNA and the amino acids that make up a protein.  Each tRNA attaches to a particular amino acid and carries it to the ribosome.  The tRNA adds its amino acid to the growing polypeptide chain.  Each tRNA could attach to only one type of amino acid.  The complex of tRNA plus its amino acid can be written in abbreviated form by adding a three-letter superscript representing the amino acid to the term tRNA.  For example, a tRNA that attaches to the amino acid alanine is written as tRNAAla. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli (2) Transfer RNA processing Associate Professor of Biochemistry and Molecular Genetics  The structure of tRNA:  A unique feature of tRNA is the presence of rare modified bases.  All RNAs have the four standard bases (adenine, cytosine, guanine, and uracil).  tRNAs have additional bases, including ribothymidine and pseudouridine.  Dr. Rami Elshazli (2) Transfer RNA processing Associate Professor of Biochemistry and Molecular Genetics  The structure of tRNA:  It is synthesized in the nucleus by transcription using RNA polymerase III enzyme, then it passes to the cytoplasm.  There are about 31 tRNA (some amino acids have more than one tRNA).  Some of the nucleotides in a tRNA are complementary to each other and form intramolecular hydrogen bonds.  As a result, each tRNA has a cloverleaf structure, having 2 free ends, and 3 loops.  The 2 free ends:  One free end (3’ terminus) = acceptor arm which ends by a specific sequence formed of CCA.  The specific activated amino acid is connected to the 3’ OH terminus by an ester link, (charged tRNA), while when tRNA is not bound to its specific activated amino acid, it is called uncharged tRNA.  (2) Transfer RNA processing  At the other end of the tRNA is a set of  The 2 free ends: three nucleotides  One free end (5’ phosphate terminus) which ends that make up the by a base guanine (over 85% of tRNA) or base anticodon. cytosine (less than 15% of tRNA).  It pairs with the  The 3 loops: corresponding codon  The anticodon loop: on the mRNA.  Central loop which is a distant from the free end carrying the amino acid.  It contains 3 bases responsible for recognition, and complementary base pairing with the 3 bases on the genetic codon of mRNA.  D loop which contains the unusual base dihydrouracil.  T loop which contains the ribothymidine and pseudouridine bases. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Processing of transfer RNA Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  After synthesis, the pre-tRNA undergoes several processing steps:  Capping:  In eukaryotes, a 5' cap is added, though this is less common for tRNA than for mRNA.  Cleavage:  The pre-tRNA is cleaved at both the 5' and 3' ends to remove extra sequences.  3' Addition of CCA:  The sequence CCA is added to the 3' end, which is essential for amino acid attachment.  Splicing:  In some cases, introns are removed, and exons are joined.  Base Modifications:  Various modifications occur to enhance the stability and function of the tRNA.  Dr. Rami Elshazli (2) Transfer RNA processing Associate Professor of Biochemistry and Molecular Genetics  Consider the mRNA codon 5′-GGC-3′, which is translated as the amino acid glycine.  The tRNA that base-pairs with this codon by hydrogen bonding has 3′-CCG-5′ as its anticodon and carries glycine at its other end.  Dr. Rami Elshazli (2) Transfer RNA processing Associate Professor of Biochemistry and Molecular Genetics  A tRNA that binds to an mRNA codon specifying a particular amino acid to the ribosome.  The correct matching up of tRNA and amino acid is carried out by a family of related enzymes called aminoacyl-tRNA synthetases.  The active site of each type of aminoacyl-tRNA synthetase fits only a specific combination of amino acid + tRNA.  There are 20 different synthetases; each synthetase is able to bind to all the different tRNAs that code for its particular amino acid.  The synthetase catalyzes the covalent attachment of the amino acid to its tRNA in a process driven by the hydrolysis of ATP.  The resulting aminoacyl tRNA, also called a charged tRNA, is released from the enzyme and is then available to deliver its amino acid to a growing polypeptide chain on a ribosome.  (2) Transfer RNA processing Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The second molecular recognition is the pairing of the tRNA anticodon with the appropriate mRNA codon.  If one tRNA existed for each mRNA codon specifying an amino acid, there would be 61 tRNAs.  There are only about 45 bacterial tRNAs, signifying that some tRNAs bind to more than one codon.  The nucleotide base U at the 5′ end of a tRNA anticodon can pair with either A or G in the third position (at the 3′ end) of an mRNA codon.  The flexible base pairing at this codon position is called wobble. ‫اﻟﺗﻣﺎﯾل واﻟﺗﻐﯾر‬  Wobble explains why the synonymous codons for a given amino acid differ in their third nucleotide base.  A tRNA with the anticodon 3′-UCU-5′ can base-pair with either the mRNA codon 5′-AGA-3′ or 5′-AGG-3′, both of which code for arginine.  (3) Ribosomal RNA processing Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Within ribosomes, the genetic instructions contained in mRNA are translated into the amino acid sequences of polypeptides.  Ribosomes are complex organelles, each consisting of different proteins and RNA molecules.  A functional ribosome consists of two subunits, a large ribosomal subunit and a small ribosomal subunit.  The sizes of the ribosomes and their RNA components are given in Svedberg (S) units.  It is a measure of how rapidly an object sediments in a centrifugal field.  A precursor RNA molecule is methylated in several places and then cleaved and trimmed to produce the mature rRNAs that make up the ribosome.  In eukaryotes, small nucleolar RNAs (snoRNAs) help to cleave and modify rRNA and assemble the processed rRNA into a mature ribosome.  Dr. Rami Elshazli (3) Ribosomal RNA processing Associate Professor of Biochemistry and Molecular Genetics Composition of ribosomes in prokaryotes and eukaryotic cells Cell type Ribosomal size Subunit rRNA component Proteins Large 50S 23S + 5S 31 Prokaryotic 70S Small 30S 16S 21 Large 60S 28S + 5.8S + 5S 49 Eukaryotic 80S Small 40S 18S 33  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics