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A20 Biochemical Society Transactions (200 I) Volume 30, Part I F12 Assembly and function of protein-RNA sub-complexes of the GI mammalian signal recognition particle S.Cusa&, 0.Weichenrieder, K. Wild, I. Sinning and K. Strub EMBL Grenoble Outstation, BP181,38042 Grenoble Cedex 9, France dementia D.A. Lomas,A.Lourbakos, S-A.Cumming and D.Belorgey Respiratory Medicine Unit, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 2 X l : UK The signal recognition particle (SRP) is a conserved ribonucleoproteia complex that mediates co-translational targeting of signal peptide bearing secretory and membrane proteins to cellular membranes. The mammalian SRP comprises the 300 nucleotide 7SL SRP RNA complexed with six proteins. The SRP14/9 heterodimer binds to the 3’ and 5’ extremities of SRP RNA forming the so-called Ah-domain, which functions to retard or arrest translation while the docking process occurs. The SRP68/72 heterodimer as well as SRP19 and SRP54 bind to the central sequences of the RNA and constitute the S-domain of SRP. SRP19 is an assembly protein which must bind to S-domain RNA before SRP54. SRP54, a GTPase, binds the RNA as well as the signal peptide and also interacts with the SRP receptor. The crystallographic structure of the Ah-domain of human SRP will be presented and its implications for the assembly of this sub-domain and its function in elongation arrest will be discussed. A second structure, of SRP19 bound to its primary binding site on SRP RNA with includes a conserved GNAR tetraloop, will also be presented. This structure gives insight into the folding of the S-domain and the requirement for prior binding of SRP19 before that of SRP54. Alpha-1-antitrypsin functions as a mousetrap to inhibit its target proteinase neutrophil elastase. The common severe Z deficiency variant (Glu342Lys) destabilises the mousetrap to allow a sequential interaction between the reactive centre loop of one molecule and Psheet A of another. These reactive loop-P-sheet A polymers accumulate within hepatocytes to form inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and hepatocelM a r carcinoma. Loop-sheet polymerisation also underlies the plasma deficiency seen with mutants of other members of the serine proteinase inhibitor (serpin) superfamily. In particular deficiency mutants of antithrombin, C1-inhibitor and al-antichymotrypsin have been demonstrated to form inactive polymers in association with thrombosis, angio-oedema and chronic obstructive pulmonary disease respectively. Moreover we have recently shown that the same process in a neurone specific protein, neuroserpin, underlies a novel inclusion body dementia. Our understanding of the structural basis of polymerisation has allowed the development of strategies to prevent the aberrant protein-protein interaction in vitro. This must now be achieved in vivo if we are to treat the associated clinical syndromes. F13 Regulatory roles for the ribosome in protein targeting to the G2 endoplasmic reticulum M.Pool, T. Fulgal, I. Sinning1, B. Dobberstein ZMBH, INF 282, 69111, Heidelberg, Germany, ‘BZH, INF 328, 69120, Heidelberg Proteins destined for secretion are synthesised with an N-terminal signal sequence which targets them to the endoplasmic reticulum (ER) membrane. Targeting occurs co-translationally and is catalysed by the signal recognition particle (SRP), together with the SRP receptor (SR), a heterodimeric intergral ER membrane protein. The targeting reaction is controlled by the action of three GTPases: SRP54 and the a and P subunits of SR. We have identified the ribosome as a regulator of two of these GTPases. In the first step of targeting, a ribosomal component, probably L35, activates SRP54 by increasing its affinity for GTP. The ribosomeSRP complex is then targeted to the ER by the interaction of SRP54 with SRa, inducing high affinity GTP binding to both proteins. Release of the signal sequence from SRP requires the presence of the translocon. This is sensed via the third GTPase SRP. In the absence of the translocon, SRP is stabilized in the nucleotide-free state by a 21 kDa ribosomal protein. Binding of the ribosome to the translocon destabilizes this interaction, allowing GTP to enter SRP which in turn triggers signal sequence release. The ribosome thereby regulates SRP, such that signal sequence release is coordinated with the presence of the translocon. 0 2002 Biochemical Society Hypersensitive mouse traps, al-antitrypsin deficiency and Gene regulation of the serine proteinase inhibitors a 1-antitrypsin and a 1-antichymotrypsin N.A.Kalsheker,K. Morgan,S.Morley Division of Clinical Cbemistry,Scbool of Clinical Laboratory Sciences, University Hospital, Queens Medical Centre, Nottingham NG7 2UH The serine proteinase inhibitors (serpins) are a superfamily of proteins with a diverse set of functions including the control of blood coagulation, complement activation, programmed cell death and development.The most abundant serpins in human plasma are al-antitrypsin (AAT) and al-antichymotrypsin (ACT). During inflammation, circulating levels can increase by up to three-fold for AAT and five-fold for ACT. The major site for increased synthesis is the liver. Other tissues, such as the lung, are also capable of synthesis and expression can be increased up to 100-fold by cytokines. For AAT there is a tissue-specific promoter for the liver and an alternative promoter for other tissues. The tissue-specific transcription factors H N F - l a and HNF-4 acting synergistically regulate basal expression. An enhancer in the 3’ flanking sequence modulates cytokine induced expression by interleukin-6 and oncostatin-M. There is probably a single promoter for ACT. Microcell hybrid transfection studies have shown that a sequence containing about 15 kb of 5’ flanking sequence is sufficient to allow stable expression of AAT in a position-independent manner. These studies should facilitate fine mapping of the elements required for expression.